Single-chain antigen-binding proteins capable of glycosylation, production and uses thereof

ABSTRACT

The present invention relates to single-chain antigen-binding molecules capable of glycosylation. Compositions of, genetic constructions coding for, and methods for producing monovalent and multivalent single-chain antigen-binding molecules capable of glycosylation are described and claimed. Composition of, genetic constructions coding for, and methods for producing glycosylated monovalent and multivalent single-chain antigen-binding molecules capable of polyalkylene oxide conjugation are described and claimed. The invention also relates to methods for producing a polypeptide having increased glycosylation and the polypeptide produced by the described method. Uses resulting from the multifunctionality of a glycosylated/polyalkylene oxide conjugated antigen-binding protein are also described and claimed.

The present application claims benefit of the filing date of U.S.Provisional Appl. No. 60/044,449, filed Apr. 30, 1997, U.S. ProvisionalAppl. No. 60/063,074, filed Oct. 27, 1997, and U.S. Provisional Appl.No. 60/067,341, filed Dec. 2, 1997, each of which disclosure isincorporated herein in entirety by reference. The present applicationalso claims benefit of the filing date of U.S. Provisional Appl. No.60/050,472, filed Jun. 23, 1997, which disclosure is incorporated hereinin entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to single-chain antigen-bindingmolecules capable of glycosylation. More specifically, the inventionrelates to antigen-binding proteins having Asn-linked glycosylationsites capable of attaching a carbohydrate moiety. The invention alsorelates to multivalent antigen-binding molecules capable ofglycosylation. The invention further relates to glycosylatedantigen-binding molecules capable of polyalkylene oxide conjugation.Compositions of, genetic constructions for, methods of use, and methodsfor producing glycosylated antigen-binding proteins capable ofpolyalkylene oxide conjugation are disclosed. The invention also relatesto methods for producing a polypeptide having increased glycosylationand the polypeptide produced by the described methods.

2. Description of the Background Art

Antibodies are proteins generated by the immune system to provide aspecific molecule capable of complexing with an invading molecule,termed an antigen. Natural antibodies have two identical antigen-bindingsites, both of which are specific to a particular antigen. The antibodymolecule “recognizes ” the antigen by complexing its antigen-bindingsites with areas of the antigen termed epitopes. The epitopes fit intothe conformational architecture of the antigen-binding sites of theantibody, enabling the antibody to bind to the antigen.

The IgG antibody, e.g., is composed of two identical heavy and twoidentical light polypeptide chains, held together by interchaindisulfide bonds. The remainder of this discussion on antibodies willrefer only to one pair of light/heavy chains, as each light/heavy pairis identical. Each individual light and heavy chain folds into regionsof approximately 110 amino acids, assuming a conserved three-dimensionalconformation. The light chain comprises one variable region (V_(L)) andone constant region (C_(L)), while the heavy chain comprises onevariable region (V_(H)) and three constant regions (C_(H)1, C_(H)2 andC_(H)3). Pairs of regions associate to form discrete structures. Inparticular, the light and heavy chain variable regions associate to forman “Fv ” area which contains the antigen-binding site.

Recent advances in immunobiology, recombinant DNA technology, andcomputer science have allowed the creation of single polypeptide chainmolecules that bind antigen. These single-chain antigen-bindingmolecules (“SCA”) or single-chain variable fragments of antibodies(“sFv”) incorporate a linker polypeptide to bridge the individualvariable regions, V_(L) and V_(H), into a single polypeptide chain. Adescription of the theory and production of single-chain antigen-bindingproteins is found in Ladner et al., U.S. Pat. Nos. 4,946,778, 5,260,203,5,455,030 and 5,518,889. The single-chain antigen-binding proteinsproduced under the process recited in the above U.S. patents havebinding specificity and affinity substantially similar to that of thecorresponding Fab fragment. A computer-assisted method for linker designis described more particularly in Ladner et al., U.S. Pat. Nos.4,704,692 and 4,881,175, and WO 94/12520.

The in vivo properties of SCA polypeptides are different from MAbs andantibody fragments. Due to their small size, SCA polypeptides clear morerapidly from the blood and penetrate more rapidly into tissues (Milenic,D. E. et al., Cancer Research 51:6363-6371 (1991); Colcher et al., J.Natl. Cancer Inst. 82:1191 (1990); Yokota et al., Cancer Research52:3402 (1992)). Due to lack of constant regions, SCA polypeptides arenot retained in tissues such as the liver and kidneys. Due to the rapidclearance and lack of constant regions, SCA polypeptides will have lowimmunogenicity. Thus, SCA polypeptides have applications in cancerdiagnosis and therapy, where rapid tissue penetration and clearance, andease of microbial production are advantageous.

A multivalent antigen-binding protein has more than one antigen-bindingsite. A multivalent antigen-binding protein comprises two or moresingle-chain protein molecules. Enhanced binding activity, di- andmulti-specific binding, and other novel uses of multivalentantigen-binding proteins have been demonstrated. See, Whitlow, M., etal., Protein Engng. 7:1017-1026 (1994); Hoogenboom, H. R., NatureBiotech. 15:125-126 (1997); and WO 93/11161.

Carbohydrate modifications of proteins fall into three generalcategories: N-linked (or Asn-linked) modification of asparagine,O-linked modification of serine or threonine andglycosyl-phosphatidylinositol derivation of the C-terminus carboxylgroup. Each of these transformations is catalyzed by one or more enzymeswhich demonstrate different peptide sequence requirements and reactionspecificities. N-linked glycosylation is catalyzed by a single enzyme,oligosaccharyl transferase (OT), and involves the co-translationaltransfer of a lipid-linked tetradecasaccharide (GlcNAc₂-Man₉-Glc₃) to anasparagine side chain within a nascent polypeptide (see, Imperiali, B.and Hendrickson, T. L., Bioorganic & Med. Chem. 3:1565-1578 (1995)). Theasparagine residue must reside within the tripeptide N-linkedglycosylation consensus sequence Asn-Xaa-Thr/Ser (NXT/S), where Xaa canbe any of the 20 natural amino acids except proline.

A natural N-linked glycosylation sequence (Asn-Val-Thr) at amino acidpositions 18-20 (Kabat's numbering) was identified in the framework-1(FR-1) region of the light chain variable domain of a murine anti-B celllymphoma antibody, LL-2 (Leung, S.-o. et al., J. Immunol. 154:5919-5926(1995)). By a single Arg to Asn mutation, an N-linked glycosylationsequence similar to that of LL-2 was introduced in the FR-1 segment of anonglycosylated, humanized anti-carcinoembryonic Ag (CEA) Ab, MN-14(Leung, S.-O. et al., J. Immunol. 154:5919-5926 (1995), which disclosureis incorporated herein by reference).

An sFv having a C-terminus that has cross-linking means by disulfidebonds at cysteine residues has been reported (Huston et al., U.S. Pat.No. 5,534,254). A monoclonal antibody has also been reported that iscovalently bound to a diagnostic or therapeutic agent through acarbohydrate moiety at an Asn-linked glycosylation site at about aminoacid position 18 of the V_(L) region (Hansen et al., U.S. Pat. No.5,443,953). Binding studies of an anti-dextran antibody that isAsn-linked glycosylated in the V_(H) chain have been performed whichshow that slight changes in the position of the Asn-linked carbohydratemoiety in the V_(H) region result in substantially different effects onantigen binding (Wright et al., EMBO J. 10:2717-2723 (1991)). It hasalso been shown that glycosylation at position 19 within the V_(H)region of an sFv enhanced expression of the overall amount of sFvintracellularly, of which approximately half was glycosylated (Greenman,J., et al., J. Immunol. Methods 194:169-180 (1996)), and enhancedsynthesis and secretion of the glycosylated sFv over the nonglycosylatedsFv (Jost, C. R., et al., J. Biol. Chem. 269:26267-26273 (1994)). Co etal., U.S. Pat. No. 5,714,350, relates to increasing binding affinity ofan antibody by eliminating a glycosylation site.

The covalent attachment of strands of a polyalkylene glycol orpolyalkylene oxide to a polypeptide molecule is disclosed in U.S. Pat.No. 4,179,337 to Davis et al., as well as in Abuchowski and Davis“Enzymes as Drugs,” Holcenberg and Roberts, Eds., pp. 367-383, JohnWiley and Sons, New York (1981), and Zalipsky et al., WO 92/16555. Thesereferences disclosed that proteins and enzymes modified withpolyethylene glycols have reduced immunogenicity and antigenicity andhave longer lifetimes in the bloodstream, compared to the parentcompounds. The resultant beneficial properties of the chemicallymodified conjugates are very useful in a variety of therapeuticapplications.

To effect covalent attachment of polyethylene glycol (PEG) and similarpoly(alkylene oxides) to a molecule, the hydroxyl end groups of thepolymer must first be converted into reactive functional groups. Thisprocess is frequently referred to as “activation” and the product iscalled “activated PEG.”

Hydrazides readily form relatively stable hydrazone linkages bycondensation with aldehydes and ketones (Andresz, H. et al., Makromol.Chem. 179:301 (1978)). This property has been used extensively formodification of glycoproteins through oxidized oligosaccharide moieties(Wilchek, M. & Bayer, E. A., Meth. Enzymol. 138:429 (1987)).

Activated PEG-hydrazide allows it to react with an aldehyde group.Aldehyde is normally absent on the polypeptide chain of a protein.However, if a protein contains carbohydrate moieties, then thecarbohydrate can be activated to provide a reactive aldehyde group byoxidation of the sugar ring such as mannose. Methods for activation ofimmunoconjugates are described in Sela et al., Immunoconjugates, Vogeled., Oxford University Press (1987). In this way, PEG-hydrazide can beconjugated covalently to the protein via the carbohydrate structure.Zalipsky, S., et al., WO 92/16555, describes PAO covalently bonded to anoxidized carbohydrate moiety of the glycopolypeptide by a linkagecontaining a hydrazide or hydrazone functional group bound to thepolymer. The oxidation of the carbohydrate moiety produces reactivealdehydes. The hydrazone linkage is formed by reacting an acyl hydrazinederivative of the polymer containing the peptide sequence with thesealdehyde groups.

The prior art has activated the hydroxyl group of PEG with cyanuricchloride and the resulting compound is then coupled with proteins(Abuchowski et al., J. Biol. Chem. 252:3578 (1977); Abuchowski & Davis,supra (1981)). However, there are disadvantages in using this method,such as the toxicity of cyanuric chloride and its non-specificreactivity for proteins having functional groups other than amines, suchas free essential cysteine or tyrosine residues.

In order to overcome these and other disadvantages, alternativeactivated PEGs, such as succinimidyl succinate derivatives of PEG(“SS-PEG”), have been introduced (Abuchowski et al., Cancer Biochem.Biophys. 7:175-186 (1984)). It reacts quickly with proteins (30 minutes)under mild conditions yielding active yet extensively modifiedconjugates.

Zalipsky, in U.S. Pat. No.5,122,614, disclosed poly(ethyleneglycol)-N-succinimide carbonate and its preparation. This form of thepolymer was said to react readily with the amino groups of proteins, aswell as low molecular weight peptides and other materials that containfree amino groups.

Other linkages between the amino groups of the protein, and the PEG arealso known in the art, such as urethane linkages (Veronese et al., Appl.Biochem. Biotechnol. 11:141-152 (1985)), carbamate linkages (Beauchampet al., Analyt. Biochem. 131:25-33 (1983)), and others.

Polyalkylene oxide modification of sFvs is disclosed in U.S. ProvisionalPatent Application No. 60/050,472, filed Jun. 23, 1997, which disclosureis incorporated herein by reference.

The activated polymers can also be reacted with a therapeutic agenthaving nucleophilic functional groups that serve as attachment sites.One nucleophilic functional group commonly used as an attachment site isthe ε-amino groups of lysines. Free carboxylic acid groups, suitablyactivated carbonyl groups, oxidized carbohydrate moieties and mercaptogroups have also been used as attachment sites.

Conjugation of poly(ethylene glycol) or poly(alkylene oxide) with smallorganic molecules is described in Greenwald, R. B., Exp. Opin. Ther.Patents 7:601-609 (1997), Enzon Inc., WO 95/11020, and Enzon Inc., WO96/23794, which disclosures are all incorporated herein by reference.Compositions based on the use of various linker groups between the PEGballast and the active drug are described in WO 96/23794.

SUMMARY OF THE INVENTION

The invention is directed to a single-chain antigen-binding polypeptidecapable of glycosylation, comprising:

(a) a first polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and(b) into a single chain polypeptide having an antigen binding site,

wherein the single-chain antigen-binding polypeptide has at least onetripeptide Asn-linked glycosylation sequence comprising Asn-Xaa-Yaa,wherein Xaa is an amino acid other than proline and Yaa is threonine orserine, wherein the tripeptide glycosylation sequence is capable ofattaching a carbohydrate moiety at the Asn residue located at a positionselected from the group consisting of (i) the amino acid position 11,12, 13, 14 or 15 of the light chain variable region; (ii) the amino acidposition 77, 78 or 79 of the light chain variable region; (iii) theamino acid position 11, 12, 13, 14 or 15 of the heavy chain variableregion; (iv) the amino acid position 82B, 82C or 83 of the heavy chainvariable region; (v) any amino acid position of the peptide linker; (vi)adjacent to the C-terminus of the second polypeptide (b); and (vii)combinations thereof, wherein the glycosylated single-chainantigen-binding polypeptide is capable of binding an antigen.

The invention is further directed to a polynucleotide encoding asingle-chain antigen-binding polypeptide capable of glycosylation,comprising:

(a) a first polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and(b) into a single chain polypeptide having an antigen binding site,

wherein the single-chain antigen-binding polypeptide has at least onetripeptide Asn-linked glycosylation sequence comprising Asn-Xaa-Yaa,wherein Xaa is an amino acid other than proline and Yaa is threonine orserine, wherein the tripeptide glycosylation sequence is capable ofattaching a carbohydrate moiety at the Asn residue located at a positionselected from the group consisting of (i) the amino acid position 11,12, 13, 14 or 15 of the light chain variable region; (ii) the amino acidposition 77, 78 or 79 of the light chain variable region; (iii) theamino acid position 11, 12, 13, 14 or 15 of the heavy chain variableregion; (iv) the amino acid position 82B, 82C or 83 of the heavy chainvariable region; (v) any amino acid position of the peptide linker; (vi)adjacent to the C-terminus of the second polypeptide (b); and (vii)combinations thereof, wherein the glycosylated single-chainantigen-binding polypeptide is capable of binding an antigen.

The polynucleotide may be DNA or RNA.

The invention is directed to a replicable cloning or expression vehiclecomprising the above described DNA sequence. The invention is alsodirected to such vehicle which is a plasmid. The invention is furtherdirected to a host cell transformed with the above described DNA. Thehost cell may be a bacterial cell, a yeast cell or other fungal cell, aninsect cell or a mammalian cell line. A preferred host is Pichiapastoris.

The invention is directed to a method of producing a single-chainantigen-binding polypeptide capable of glycosylation, comprising:

(a) providing a first polynucleotide encoding a first polypeptidecomprising the antigen binding portion of the variable region of anantibody heavy or light chain;

(b) providing a second polynucleotide encoding a second polypeptidecomprising the antigen binding portion of the variable region of anantibody heavy or light chain; and

(c) linking the first and second polynucleotides (a) and (b) with athird polynucleotide encoding a peptide linker into a fourthpolynucleotide encoding a single chain polypeptide having an antigenbinding site,

wherein the single-chain antigen-binding polypeptide has at least onetripeptide Asn-linked glycosylation sequence comprising Asn-Xaa-Yaa,wherein Xaa is an amino acid other than proline and Yaa is threonine orserine, wherein the tripeptide glycosylation sequence is capable ofattaching a carbohydrate moiety at the Asn residue located at a positionselected from the group consisting of (i) the amino acid position 11,12, 13, 14 or 15 of the light chain variable region; (ii) the amino acidposition 77, 78 or 79 of the light chain variable region; (iii) theamino acid position 11, 12, 13, 14 or 15 of the heavy chain variableregion; (iv) the amino acid position 82B, 82C or 83 of the heavy chainvariable region; (v) any amino acid position of the peptide linker; (vi)adjacent to the C-terminus of the second polypeptide (b); and (vii)combinations thereof, wherein the glycosylated single-chainantigen-binding polypeptide is capable of binding an antigen; and

(d) expressing the single-chain antigen-binding polypeptide of (c) inthe host, thereby producing a single-chain antigen-binding polypeptidecapable of glycosylation.

In the method as according to the invention, the host cell is capable ofcatalyzing glycosylation. The host cell is a plant cell, a bacterialcell, a yeast cell or other fungal cell, an insect cell or a mammaliancell line. A preferred host cell is Pichia pastoris.

The invention is further directed to a multivalent single-chainantigen-binding protein, comprising two or more single-chainantigen-binding polypeptides, each single-chain antigen-bindingpolypeptide comprising:

(a) a first polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and(b) into a single chain polypeptide having an antigen binding site,wherein the single-chain antigen-binding polypeptide has at least onetripeptide Asn-linked glycosylation sequence comprising Asn-Xaa-Yaa,wherein Xaa is an amino acid other than proline and Yaa is threonine orserine, wherein the tripeptide glycosylation sequence is capable ofattaching a carbohydrate moiety at the Asn residue located at a positionselected from the group consisting of (i) the amino acid position 11,12, 13, 14 or 15 of the light chain variable region; (ii) the amino acidposition 77, 78 or 79 of the light chain variable region; (iii) theamino acid position 11, 12, 13, 14 or 15 of the heavy chain variableregion; (iv) the amino acid position 82B, 82C or 83 of the heavy chainvariable region; (v) any amino acid position of the peptide linker; (vi)adjacent to the C-termninus of the second polypeptide (b); and (vii)combinations thereof, wherein the glycosylated single-chainantigen-binding polypeptide is capable of binding an antigen.

In the above described embodiments of the invention, the tripeptideglycosylation sequence may be capable of attaching a carbohydrate moietyat the Asn residue located at a position selected from the groupconsisting of (i′) the amino acid position 12 of the light chainvariable region; (ii′) the amino acid position 77 of the light chainvariable region; (iii′) the amino acid position 13 of the heavy chainvariable region; (iv′) the amino acid position 82B of the heavy chainvariable region; (v′) the amino acid position 2 of the peptide linker;(vi′) adjacent to the C-terminus of the second polypeptide (b); and(vii′) combinations thereof, wherein the glycosylated single-chainantigen-binding polypeptide is capable of binding an antigen.

In the above described embodiments of the invention, at least onesingle-chain antigen-binding polypeptide may have at least twotripeptide glycosylation sequences in tandem such that the Asn residuesare separated by two amino acid residues and/or at least one set of twooverlapping tripeptide glycosylation sequences such that the Asnresidues are adjacent. At least one single-chain antigen-bindingpolypeptide may have three tripeptide glycosylation sequences in tandem.At least one single-chain antigen-binding polypeptide may have at leasttwo sets of two tandem tripeptide glycosylation sequences and at leasttwo sets of two overlapping tripeptide glycosylation sequences.

Also in the above described embodiments of the invention, the Asnresidue of the tripeptide glycosylation sequence may be attached to acarbohydrate moiety. The carbohydrate moiety may further be conjugatedto polyalkylene oxide. The carbohydrate and/or polyalkylene moieties maybe conjugated to one or plurality of peptide, lipid, nucleic acid, drug,toxin, chelator, boron addend or detectable label molecule(s). Thecarbohydrate and/or polyalkylene oxide moieties may be conjugated to acarrier having one or plurality of peptide, lipid, nucleic acid, drug,toxin, chelator, boron addend or detectable label molecule(s) bound tothe carrier.

In the above described embodiments of the invention, the C-terminus ofthe second polypeptide (b) may be the native C-terminus of the secondpolypeptide (b). In the alternative, the C-terminus of the secondpolypeptide (b) may comprise a deletion of one or plurality of aminoacid residue(s), such that the remaining N-terminus amino acid residuesof the second polypeptide are sufficient for the glycosylatedpolypeptide to be capable of binding an antigen. In the alternative, theC-terminus of the second polypeptide may comprise an addition of one orplurality of amino acid residue(s), such that the glycosylatedpolypeptide is capable of binding an antigen. In one embodiment, the Asnresidue of the glycosylation sequence may be located adjacent to any ofthe above mentioned C-terminus of the second polypeptide and theglycosylation sequence may be followed by at least one amino acidresidue. In the alternative, the glycosylation sequence may be followedby two, three, four or five amino acid residues.

In a preferred embodiment of the invention, the first polypeptide (a)may comprise the antigen binding portion of the variable region of anantibody light chain and the second polypeptide (b) comprises theantigen binding portion of the variable region of an antibody heavychain.

The invention is also directed to a method of detecting an antigensuspected of being in a sample, comprising:

(a) contacting the sample with the glycosylated polypeptide ormultivalent protein of the invention, wherein the carbohydrate moiety isconjugated to one or plurality of detectable label molecule(s), orconjugated to a carrier having one or plurality of detectable labelmolecule(s) bound to the carrier; and

(b) detecting whether the glycosylated single-chain antigen-bindingpolypeptide has bound to the antigen.

The invention is further directed to a method of imaging the internalstructure of an animal, comprising administering to the animal aneffective amount of the glycosylated polypeptide or multivalent proteinof the invention, wherein the carbohydrate moiety is conjugated to oneor plurality of detectable label or chelator molecule(s), or conjugatedto a carrier having one or plurality of detectable label or chelatormolecule(s) bound to the carrier, and measuring detectable radiationassociated with the animal. Animal includes human and nonhuman.

The invention is also directed to a method for treating a targeteddisease, comprising administering an effective amount of a compositioncomprising the glycosylated polypeptide or multivalent protein of theinvention and a pharmaceutically acceptable carrier vehicle, wherein thecarbohydrate moiety is conjugated to one or plurality of peptide, lipid,nucleic acid, drug, toxin, boron addend or radioisotope molecule(s), orconjugated to a carrier having one or plurality of drug, toxin, boronaddend or radioisotope molecule(s) bound to the carrier.

The above described methods may be facilitated with the glycosylatedpolypeptide or multivalent protein of the invention, which is conjugatedto polyalkylene oxide which may also be conjugated to one or pluralityof peptide, lipid, nucleic acid, drug, toxin, chelator, boron addend ordetectable label molecule(s).

The invention also relates to (1) a method of producing a polypeptidehaving increased glycosylation, comprising: (a) providing to apolynucleotide encoding the polypeptide at least two tripeptideAsn-linked glycosylation sequences, wherein each tripeptideglycosylation sequence comprises Asn-Xaa-Yaa, wherein Xaa is an aminoacid other than proline and Yaa is threonine or serine, and wherein thetripeptide glycosylation sequences are in tandem such that the Asnresidues are separated by two amino acid residues; and (b) expressingthe polynucleotide in a host cell capable of attaching a carbohydratemoiety at the Asn residues, and (2) a polypeptide having increasedglycosylation produced by the described process.

The invention further relates to (1) a method of producing a polypeptidehaving increased glycosylation, comprising: (a) providing to apolynucleotide encoding the polypeptide at least one set of twotripeptide Asn-linked glycosylation sequences, wherein each tripeptideglycosylation sequence comprises Asn-Xaa-Yaa, wherein Xaa is an aminoacid other than proline and Yaa is threonine or serine, and wherein thetwo tripeptide glycosylation sequences overlap such that the Asnresidues are adjacent; and (b) expressing the polynucleotide in a hostcell capable of attaching a carbohydrate moiety at the Asn residues, and(2) a polypeptide having increased glycosylation produced by thedescribed process.

The invention also relates to (1) a method of producing a polypeptidehaving increased glycosylation, comprising: (a) providing to apolynucleotide encoding the polypeptide at least two tripeptideAsn-linked glycosylation sequences, wherein each tripeptideglycosylation sequence comprises Asn-Xaa-Yaa, wherein Xaa is an aminoacid other than proline and Yaa is threonine or serine, and wherein thetripeptide glycosylation sequences are in tandem such that the Asnresidues are separated by two amino acid residues; (b) providing to thepolynucleotide at least one set of two tripeptide Asn-linkedglycosylation sequences, wherein the two tripeptide glycosylationsequences overlap such that the Asn residues are adjacent; and (c)expressing the polynucleotide in a host cell capable of attaching acarbohydrate moiety at the Asn residues, and (2) a polypeptide havingincreased glycosylation produced by the described process.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B . DNA sequence (SEQ ID NO:1) and translated proteinsequence (SEQ ID NO:2) of CC49/218 SCA with engineered N-linkedglycosylation sites indicated. The variable light (V_(L)) and variableheavy (V_(H)) chains are indicated. Restriction sites are underlined andnamed. CDR sequences are double underlined. The 218 linker is underlinedand named. Proposed tripeptide N-linked glycosylation sites areunderlined. The mutations made to generate the N-linked glycosylationsites are shown in parenthesis underneath the proposed glycosylationsites and the site of oligosaccharide attachment is denoted under themutated sequence by * and numbered. The C-terminal extension forglycosylation is shown in parenthesis.

FIG. 2A and 2B. Binding of serial dilutions of parent CC49/218 SCAobtained from E. coli GX9251 (FIG. 2A) or P. pastoris EN225 (FIG. 2B) toimmobilized bovine submaxillary mucin in ELISA. The purified CC49/218SCA from E. coli (100 μg/ml) and the unpurified culture supernatant fromP. pastoris EN225 (˜50 μg/ml SCA) were assayed for direct binding ofantigen as described in Materials and Methods. Absorbance at 405 nm(A405) was measured after 10 min of PNPP substrate incubation with thealkaline phosphatase conjugated rabbit anti-mouse antibody. Two controlsare shown at the right. C1 records the absorbance of CC49/218 (3.3-folddilution) assayed with immobilized porcine submaxillary mucin. C2 showsthe background binding of induced P. pastoris host GS115 (3.3-folddilution) to immobilized bovine submaxillary mucin.

FIG. 3. Western blot analysis of CC49/218 SCA and glyco-SCA before andafter enzymatic treatment with N-acetylglucosamine specificendoglycosidases. Unpurified CC49/218 SCA from culture supernatants ofEN225, EN235 and EN236 were digested with Peptide-N-glycosidase F orEndo-glycosidase H. The samples (˜1 μg per lane) were run on a 4-20%SDS-PAGE slab gel and transferred to a nitrocellulose membrane forWestern analysis using a rabbit anti-CC49/218 SCA polyclonal antibody.Lanes 1-3, EN236; Lanes 4-6, EN235; Lanes 7-9, EN225. Lanes 1,4, and 7,Endo-glycosidase H treated; Lanes 2, 5, and 8, Peptide-N-glycosidase Ftreated; Lanes 3, 6, and 9, untreated. The flanking lane 10 (not shown)contained molecular weight markers and showed no cross reactivity to theantibody.

FIGS. 4A and 4B. Affinity chromatography of CC49/218 SCA from culturesupernatant of EN235. SDS-PAGE analysis was performed on EN235 derivedmixture of unmodified SCA and glyco-SCA following chromatography on amucin-Sepharose column as described in the “Materials and Methods”section of Example 1. The Coomassie Blue stained gel was scanned using aMolecular Dynamics Laser Scanner Model PD-SI and the area quantitationis displayed for the purified sample (FIG. 4A) and the startingsupernatant (FIG. 4B). The ratios of glycosylated SCA (peak 1) tounmodified SCA (peak 2) are 1.2 (FIG. 4A) and 1.1 (FIG. 4B).

FIGS. 5A and 5B(a-c). Lectin specific separation of glyco-SCA fromunmodified SCA by Con A Sepharose. CC49/218 SCA from EN235 culturesupernatant was incubated with molar excess of Con A Sepharose resin(Pharmacia Biotech). The unbound supernatant fraction was removed, andthe bound fraction was eluted with alpha-D-methylmannoside. SDS-PAGEanalysis was performed on 4-20% slab gels (˜1 μg per lane). TheCoomassie Blue stained gel is shown in FIG. 5A: Lane a, bound fraction;Lane b, unbound fraction; Lane c, untreated culture supernatant. The gelwas scanned as in FIGS. 4A and 4B and the results are shown in FIG.5B(a-c), where peaks 1 and 2 correspond to bands 1 and 2, respectively.

FIG. 6. Kabat consensus V_(k)I/218/V_(H)III SCA with engineeredglycosylation sites. Amino acid sequence (SEQ ID NO:3) and engineeredN-linked glycosylation sites for a consensus human SCA proteincontaining a V_(L) domain (derived from a human kappa light chainsubgroup I consensus sequence) and a V_(H) domain (derived from a humanheavy chain subgroup III consensus sequence) which are tethered by the218-linker. Amino acid assignments are according to Kabat et al.,Sequences of Proteins of Immunological Interest, pp. 108 & 331, 5th ed.,U.S. Dept. Health and Human Services, Bethesda, Md. (1991), where theassigned amino acid residue at a position is the most commonly occurringamino acid at that position. The amino acids are listed according to thestandard one letter codon and X denotes any amino acid. CDR sequencesare double underlined. The 218 linker is overlined and named. Proposedtripeptide N-linked glycosylation sites are underlined and the site ofoligosaccharide attachment is indicated by *. Proposed residue(s) changeto generate N-linked glycosylation site is in parenthesis below. TheC-terminal extension for glycosylation is shown in parenthesis. Thethree uncommon V_(K)I CDR1 positions 27D, 27E, and 27F are not shown.The V_(H)III terminal position 113 is optional and alternate SCA mayterminate at position 112. Proline residues flanking the tripeptidesequence in the +3 position are changed to alanines, as recommended bythe compilation of Gavel, Y., and von Heijne, G., Protein Engng.3:433-442 (1990).

FIG. 7. C6.5/218 SCA with engineered glycosylation sites. Amino acidsequence (SEQ ID NO:4) and engineered N-linked glycosylation sites forthe human C6.5 SCA protein containing a V_(L) domain (derived from ahuman lambda chain subgroup 1 segment) and a V_(H) domain (derived froma human heavy chain subgroup 5 segment) which are tethered by the218-linker. Amino acid assignments of the wild-type C6.5 variabledomains are according to Schier, R., et al., J. Mol. Biol. 255:28-43(1996). CDR sequences are double underlined. The 218 linker is overlinedand named. Proposed tripeptide N-linked glycosylation sites areunderlined and the site of oligosaccharide attachment is indicated by *.Proposed residue(s) change to generate N-linked glycosylation site is inparenthesis below. The C-terminal extension for glycosylation is shownin parenthesis. The V_(H) terminal position 113 is optional and analternate SCA may terminate at position 112. Proline residues flankingthe tripeptide sequence in the +3 position are changed to alanines, asrecommended by the compilation of Gavel, Y., and von Heijne, G., ProteinEngng. 3:433-442 (1990).

FIG. 8. A33/218 SCA with engineered glycosylation sites. Amino acidsequence (SEQ ID NO:5) and engineered N-linked glycosylation sites forthe murine A33 SCA protein of pGX9451 containing a mouse V_(L) domainand a mouse V_(H) domain which are tethered by the 218-linker. Aminoacid assignments conform to the numbering system of Kabat et al.,Sequences of Proteins of Immunological Interest, 5th ed., U.S. Dept.Health and Human Services, Bethesda, Md. (1991). CDR sequences aredouble underlined. The 218 linker is overlined and named. Proposedtripeptide N-linked glycosylation sites are underlined and the site ofoligosaccharide attachment is indicated by *. Proposed residue(s) changeto generate N-linked glycosylation site is in parenthesis below. TheC-terminal extension for glycosylation is shown in parenthesis.

FIG. 9. Western blot analysis of CC49/218 SCA and glyco-CC49/218 SCA,having one, two, or three glycosylation sites, before and aftertreatment with Endo-glycosidase H. Conditions were as described above.Lane 1, control P. pastoris host GS115; Lane 2, CC49/218 SCA; Lanes 3and 4, glyco-CC49/218 SCA EN236 (one glycosylation site); Lanes 5 and 6,glyco-CC49/218 SCA EN279 (two glycosylation sites); Lanes 7 and 8,glyco-CC49/218 SCA EN280 (three glycosylation sites). Lanes 4, 6, and 8,treated with Endo-glycosidase H.

FIG. 10. ELISA quantitation of mucin-binding activity by unmodified CC49SCA (E. coli CC49 and P. pastoris EN 225) and CC49 with one (EN 236),two (EN 279), or three (EN 280) glycosylation sites. The two controls,BSA and GS115 (P. pastoris host), showed very little mucin bindingactivity. =BSA; =E. coli CC49; =GS115; =EN225; =EN236; =EN279; and=EN280.

FIGS. 11A and 11B. FIG. 11A. SEC chromatography of glycosylated andPEG-modified CC49 with two tandem glycosylation sites. The glyco-CC49/2(EN279) was purified by a combination of cation exchange chromatographyand anion exchange chromatography. Conditions for PEGylation (PEGmodification) of the glycosylated CC49/2 were as described in Example 4.SEC chromatography of the reaction mixture showed the appearance of highmolecular weight peaks in addition to the low molecular weightnon-glycosylated peak which was the only peak before PEGylation. FIG.11B. SDS-PAGE analysis of the reaction mixture showed that theglycosylated and PEGylated CC49/2 was all converted to a highermolecular weight species (lane 2). Lanes 2 and 3 contained a 50/50mixture of glycosylated and non-glycosylated CC49 which was not PEGhydrazide modified. The non-glycosylated CC49/2 (Lane 3) remained at theposition corresponding to the un-modified species. This indicated thatthe reaction was specific for the carbohydrate moiety and did not affectthe SCA that contains no carbohydrate. Lanes 1 and 4 contained molecularweight standards.

FIG. 12. SDS-PAGE analysis of the fractions from size exclusionchromatography after PEGylation of glyco-CC49/2 (EN279). Lane 1,molecular weight marker; Lane 2, native unPEGylated gCC49/2(nonglycosylated and glycosylated fractions); Lane 3, low molecularweight fraction (nonglycosylated SCA); Lane 4, high molecular weightfraction (PEGylated and glycosylated single chain gCC49/2).

FIG. 13. SDS-PAGE analysis of gCC49/3 (glyco-CC49/triple sites) (EN280)and PEGylated gCC49/3. Conditions for PEGylation of the glycosylatedCC49/3 were as described in Example 5. Lane 1, native un-PEGylatedglycosylated CC49/3; Lane 2, gCC49/3-HZ5,000-PEG (highly PEGylatedfraction); Lane 3, gCC49/3-HZ5,000-PEG (less PEGylated fraction); Lane4, molecular weight marker (116.3, 97.4, 66.3, 55.4, 36.5, 31, 26.5(CC29/218-native), 21.5, 14.4, 6 kDa). Both the highly PEGylated andless PEGylated fractions of the PEGylated and glycosylated CC49/3 hadmolecular weights much higher than that of the un-PEGylated glycosylatedCC49/3.

FIG. 14. Circulation life of Glyco-SCA and PEG-Glyco-SCA. =gCC49/3(EN280); =PEG5-gCC49/3. Details are described in Example 6.

FIG. 15. Pharmacokinetics of Plasma Retention of SCA and PEG-SCA. =CC49;and =PEG20-CC49. Details are described in Example 7.

FIG. 16. Western blot of SCAs: Lanes 1 and 10, Mol. Wt. Markers; Lane 2,EN293; Lane 3, EN280; Lane 4, EN294; Lane 5, EN279; Lane 6, EN292; Lane7, EN236; Lane 8, E. coli CC49; Lane 9, GS115.

FIG. 17. Western blot of EN290 (three glycosylation sequences in linkerregion) and EN225 (CC49 parent). Mol. wt. markers are indicated (27 kDa,30 kDa, and 43 kDa).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to the discovery that glycosylated single-chainantigen-binding molecules (“SCA”) or single-chain variable fragments ofantibodies (“sFv”) have significant utility beyond that of thenonglycosylated single-chain antigen-binding proteins. In addition tomaintaining an antigen binding site, a glycosylated SCA protein has acarbohydrate moiety which acts as a second biological effector. Theoligosaccharide functions may include cellular and tissue targeting,specific binding and interactions with serum proteins, specific bindingand interactions with cell receptors, cell matrix and intracellularproteins. Accordingly, the invention is directed to monovalent andmultivalent SCA proteins capable of glycosylation, compositions ofmonovalent and multivalent glycosylated SCA proteins, methods of makingand purifying monovalent and multivalent glycosylated SCA proteins, anduses for glycosylated SCA proteins. The invention is also directed toglycosylated SCA proteins having a diagnostic or therapeutic agentcovalently attached to an Asn-linked carbohydrate moiety.

The terms “single-chain antigen-binding molecule” (SCA) or “single-chainFv” (sFv) are used interchangeably here. They are structurally definedas comprising the binding portion of a first polypeptide from thevariable region of an antibody V_(L) (or C_(H)), associated with thebinding portion of a second polypeptide from the variable region of anantibody V_(H) (or C_(L)), the two polypeptides being joined by apeptide linker linking the first and second polypeptides into a singlepolypeptide chain, such that the first polypeptide is N-terminal to thelinker and second polypeptide is C-terminal to the first polypeptide andlinker. The single polypeptide chain thus comprises a pair of variableregions connected by a polypeptide linker. The regions may associate toform a functional antigen-binding site, as in the case wherein theregions comprise a light-chain and a heavy-chain variable region pairwith appropriately paired complementarity determining regions (CDRs). Inthis case, the single-chain protein is referred to as a “single-chainantigen-binding protein” or “single-chain antigen-binding molecule.”

Single-chain Fvs can and have been constructed in several ways. EitherV_(L) is the N-terminal domain followed by the linker and V_(H) (aV_(L)-Linker-V_(H) construction) or V_(H) is the N-terminal domainfollowed by the linker and V_(L) (V_(H)-Linker-V_(L) construction). Thepreferred embodiment contains V_(L) in the N-terminal domain (see,Anand, N. N., et al., J. Biol. Chem. 266:21874-21879 (1991)).Alternatively, multiple linkers have also been used. Several types ofsFv proteins have been successfully constructed and purified, and haveshown binding affinities and specificities similar to the antibodiesfrom which they were derived.

A description of the theory and production of single-chainantigen-binding proteins is found in Ladner et al., U.S. Pat. Nos.4,946,778, 5,260,203, 5,455,030 and 5,518,889, and in Huston et al.,U.S. Pat. No. 5,091,513 (“biosynthetic antibody binding sites” (BABS)),which disclosures are all incorporated herein by reference. Thesingle-chain antigen-binding proteins produced under the process recitedin the above patents have binding specificity and affinity substantiallysimilar to that of the corresponding Fab fragment.

Typically, the Fv domains have been selected from the group ofmonoclonal antibodies known by their abbreviations in the literature as26-10, MOPC 315, 741F8, 520C9, McPC 603, D1.3, murine phOx, human phOx,RFL3.8 sTCR, 1A6, Se155-4, 18-2-3, 4-4-20, 7A4-1, B6.2, CC49, 3C2, 2c,MA-15C5/K₁₂G₀, Ox, etc. (see, Huston, J. S. et al., Proc. Natl. Acad.Sci. USA 85:5879-5883 (1988); Huston, J. S. et al., SIM News 38(4)(Supp.):11 (1988); McCartney, J. et al., ICSU Short Reports 10:114(1990); McCartney, J. E. et al., unpublished results (1990); Nedelman,M. A. et al., J. Nuclear Med. 32 (Supp.):1005 (1991); Huston, J. S. etal., In: Molecular Design and Modeling: Concepts and Applications, PartB, edited by J. J. Langone, Methods in Enzymology 203:46-88 (1991);Huston, J. S. et al., In: Advances in the Applications of MonoclonalAntibodies in Clinical Oncology, Epenetos, A. A. (Ed.), London, Chapman& Hall (1993); Bird, R. E. et al., Science 242:423-426 (1988); Bedzyk,W. D. et al., J. Biol. Chem. 265:18615-18620 (1990); Colcher, D. et al.,J. Nat. Cancer Inst. 82:1191-1197 (1990); Gibbs, R. A. et al., Proc.Natl. Acad. Sci. USA 88:4001-4004 (1991); Milenic, D. E. et al., CancerResearch 51:6363-6371 (1991); Pantoliano, M. W. et al., Biochemistry30:10117-10125 (1991); Chaudhary, V. K. et al., Nature 339:394-397(1989); Chaudhary, V. K. et al., Proc. Natl. Acad. Sci. USA 87:1066-1070(1990); Batra, J. K. et al., Biochem. Biophys. Res. Comm. 171:1-6(1990); Batra, J. K. et al., J. Biol. Chem. 265:15198-15202 (1990);Chaudhary, V. K. et al., Proc. Natl. Acad. Sci. USA 87:9491-9494 (1990);Batra, J. K. et al., Mol. Cell. Biol. 11:2200-2205 (1991); Brinkmann, U.et al., Proc. Natl. Acad Sci. USA 88:8616-8620 (1991); Seetharam, S. etal., J. Biol. Chem. 266:17376-17381 (1991); Brinkmann, U. et al., Proc.Natl. Acad. Sci. USA 89:3075-3079 (1992); Glockshuber, R. et al.,Biochemistry 29:1362-1367 (1990); Skerra, A. et al., Bio/Technol.9:273-278 (1991); Pack, P. et al., Biochemistry 31:1579-1534 (1992);Clackson, T. et al., Nature 352:624-628 (1991); Marks, J. D. et al., J.Mol. Biol. 222:581-597 (1991); Iverson, B. L. et al., Science249:659-662 (1990); Roberts, V. A. et al., Proc. Natl. Acad. Sci. USA87:6654-6658 (1990); Condra, J. H. et al., J. Biol. Chem. 265:2292-2295(1990); Laroche, Y. et al., J. Biol. Chem. 266:16343-16349 (1991);Holvoet, P. et al., J. Biol. Chem. 266:19717-19724 (1991); Anand, N. N.et al., J. Biol. Chem. 266:21874-21879 (1991); Fuchs, P. et al.,Bio/Technol. 9:1369-1372 (1991); Breitling, F. et al., Gene 104:104-153(1991); Seehaus, T. et al., Gene 114:235-237 (1992); Takkinen, K. etal., Protein Engng. 4:837-841 (1991); Dreher, M. L. et al., J. Immunol.Methods 139:197-205 (1991); Mottez, E. et al., Eur. J. Immunol.21:467-471 (1991); Traunecker, A. et al., Proc. Natl. Acad. Sci. USA88:8646-8650 (1991); Traunecker, A. et al., EMBO J. 10:3655-3659 (1991);Hoo, W. F. S. et al., Proc. Natl. Acad. Sci. USA 89:4759-4763 (1993)).

Linkers of the invention used to construct SCA polypeptides are designedto span the C-terminus of V_(L) (or neighboring site thereof) and theN-terminus of V_(H) (or neighboring site thereof) or between theC-terminus of V_(H) and the N-terminus of V_(L). The preferred length ofthe peptide linker should be from 2 to about 50 amino acids. In eachparticular case, the preferred length will depend upon the nature of thepolypeptides to be linked and the desired activity of the linked fusionpolypeptide resulting from the linkage. Generally, the linker should belong enough to allow the resulting linked fusion polypeptide to properlyfold into a conformation providing the desired biological activity.Where conformational information is available, as is the case with SCApolypeptides discussed below, the appropriate linker length may beestimated by consideration of the 3-dimensional conformation of thesubstituent polypeptides and the desired conformation of the resultinglinked fusion polypeptide. Where such information is not available, theappropriate linker length may be empirically determined by testing aseries of linked fusion polypeptides with linkers of varying lengths forthe desired biological activity. Such linkers are described in detail inWO 94/12520, which disclosure is incorporated herein by reference.

Preferred linkers used to construct SCA polypeptides have between 10 and30 amino acid residues. The linkers are designed to be flexible, and itis recommended that an underlying sequence of alternating Gly and Serresidues be used. To enhance the solubility of the linker and itsassociated single chain Fv protein, three charged residues may beincluded, two positively charged lysine residues (K) and one negativelycharged glutamic acid residue (E). Preferably, one of the lysineresidues is placed close to the N-terminus of V_(H), to replace thepositive charge lost when forming the peptide bond of the linker and theV_(H).

In addition, it has been found that linker lengths of equal to orgreater than 18 residues reduce aggregation. This becomes important athigh concentrations, when aggregation tends to become evident. Thus,linkers having 18 to 30 residues are most preferred for SCA polypeptidesin the monovalent conformation. Linker lengths of less than 10 residuesare favored for SCA in the multimer conformation.

Another property that is important in engineering an SCA polypeptide, orany other linked fusion polypeptide, is proteolytic stability. The 212linker (Pantoliano et al., Biochemistry 30:10117 (1991)) is susceptibleto proteolysis by subtilisin BPN′. The proteolytic clip in the 212linker occurs between Lys8 and Ser9 of the linker (see Table 1). Byplacing a proline at the proteolytic clip site one may be able toprotect the linker.

Table 1 shows various linkers for illustration. See also, Whitlow, M.,et al., Protein Engng. 7:1017-1026 (1994). The 217 linker contains alysine-proline pair at positions 6 and 7; the 218 linker contains thelysine-proline pair at positions 8 and 9, respectively, thus renderingthe linker less susceptible to proteolysis. The 218 linker demonstratesless aggregation, greater proteolytic stability, and the necessaryflexibility and solubility to result in a functional linker for SCAproteins.

TABLE 1 Linker Designs Linker Linker Name Reference GKSSGSGSESKS⁽³⁾ 202′Bird et al.⁽¹⁾ GSTSGSGKSSEGKG⁽⁴⁾ 212 Bedzyk et al.⁽²⁾GSTSGSGKSSEGSGSTKG⁽⁵⁾ 216 212 Experimental Derivative GSTSGKPSEGKG⁽⁶⁾217 WO 94/12520 GSTSGSGKPGSGEGSTKG⁽⁷⁾ 218 WO 94/12520 ⁽¹⁾Science 242:423(1988) ⁽²⁾J. Biol. Chem. 265:18615-18620 (1990) ⁽³⁾SEQ. ID NO. 6 ⁽⁴⁾SEQ.ID NO. 7 ⁽⁵⁾SEQ. ID NO. 8 ⁽⁶⁾SEQ. ID NO. 9 ⁽⁷⁾SEQ. ID NO. 10

A second guiding consideration in linker design is that a linker withreduced aggregation is preferable. As described above, the 18-residue216 linker shows reduced aggregation as compared to the 14-residue 212linker. The first eleven residues of the 216 linker are identical to the212 linker, including the proteolytically-susceptible peptide bondbetween Lys8 and Ser9. Thus, it is believed that the extra four residuescontribute to the lowered aggregation. Linkers with 18 or more residuesare thus most preferred.

Taking the above into consideration, a linker was designed in which aproline was substituted for serine at position 9, after Lys8, in the18-residue 216 linker. The linker is designated 218 (see Table 1). SeeWO 94/12520, which disclosure is incorporated herein by reference.

Positioning the proline at the proper place in the linker sequence toinhibit proteolysis is accomplished by determining the points ofproteolytic attack in the susceptible sequence. One of ordinary skill inthe art will know of methods of determining this point. In one method, aprotease such as subtilisin BPN′ is contacted with the candidate linker.Cleavage can then be determined by sequencing the resulting peptides,which will also reveal the cleavage point or points, if any. Anyprotease may be used, and selection will be guided by consideration ofthe environment the linker is to encounter in actual use.

The requirements for an SCA is that the linker be longer than 12 aminoacids. The preferred length of the linker in an SCA is greater than 18residues, in order to reduce aggregation, as described above.

For multivalent SCAs, the association of two or more SCAs is requiredfor their formation. Although, multivalent SCAs can be produced fromSCAs with linkers as long as 25 residues, they tend to be unstable.Holliger, P., et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993),have recently demonstrated that linkers 0 to 15 residues in lengthfacilitate the formation of divalent Fvs. See, Whitlow, M., et al.,Protein Engng. 7:1017-1026 (1994); Hoogenboom, H. R.,

Nature Biotech. 15:125-126 (1997); and WO 93/11161.

The object of the present invention is to produce an SCA having one ormore (or at least one) Asn-linked glycosylation sequence(s) such thatthe Asn-linked glycosylation site (the asparagine residue within theglycosylation sequence) is capable of attaching a carbohydrate moietyand the glycosylated polypeptide is capable of binding an antigen (i.e.,the glycosylated polypeptide's ability to bind an antigen is notdisrupted). For example, the SCA may have one, two, three, five, sevenor ten N-linked glycosylation sequence(s), but not limited to thenumbers recited. Asn-linked glycosylation, also referred to as N-linkedglycosylation, occurs when sugar residues are linked through the amidenitrogen of asparagine residues. Intracellular biosynthesis ofAsn-linked oligosaccharides occurs in both the lumen of the endoplasmicreticulum and following transport of the protein to the Golgi apparatus.Asn-linked glycosylation occurs at the following tripeptideglycosylation consensus sequence: Asn-Xaa-Yaa (Asn-Xaa-Thr/Ser; NXT/S),where Xaa may be any amino acid except proline and Yaa is serine orthreonine.

All Asn-linked oligosaccharides have a common pentasaccharide core(Man₃GlcNAc₂) originating from a common biosynthetic intermediate. Theydiffer in the number of branches and the presence of peripheral sugarssuch as fucose and sialic acid. They can be categorized according totheir branched constituents, which may consist of mannose only (highmannose N-glycans); alternating GlcNAc and Gal residues terminated byvarious sugar sequences, and with the possibility of intrachainsubstitutions of bisecting Fuc and core GlcNAc (complex N-glycans); orattributes of both high mannose and complex chains (hybrid N-glycans).See, Hounsell, E. F ed., “Glycoprotein Analysis in Biomedicine,” Methodsin Molecular Biology 14:298 (1993).

A further object of the invention is to produce monovalent andmultivalent SCAs having one or more Asn-linked glycosylationsequence(s). For multivalent SCAs, the association of two or more SCAsis required for their formation. For example, multivalent SCAs may begenerated by chemically crosslinking two SCAs with C-terminal cysteineresidues (Cumber et al., J. Immunol. 149:120-126 (1992)) and by linkingtwo SCAs with a third polypeptide linker to form a dimeric Fv (George etal., J. Cell. Biochem. 15E:127 (1991)). Details for producingmultivalent SCAs by aggregation are described in Whitlow, M., et al.,Protein Engng. 7:1017-1026 (1994). Multivalent antigen-binding fusionproteins of the invention can be made by any process, but preferablyaccording to the process for making multivalent antigen-binding proteinsset forth in WO 93/11161, which disclosure is incorporated herein byreference.

Yet a further object of the invention is to produce monovalent ormultivalent SCAs, as described above, having at least two tripeptideglycosylation sequences in tandem such that the Asn residues areseparated by two amino acid residues. Another object of the invention isto produce monovalent or multivalent SCAs, as described above, having atleast one set of two overlapping tripeptide glycosylation sequences suchthat the Asn residues are adjacent. Another object of the invention isto produce monovalent or multivalent glycosylated SCAs, as describedabove, which are conjugated to polyalkylene oxide.

Identification and Synthesis of N-linked Glycosylation Sequences

In the present invention, N-linked glycosylation sites within thetripeptide glycosylation consensus sequences may occur in the V_(L) andV_(H) regions, the C-termninus of the second polypeptide (V_(L), V_(H)or neighboring site thereof), the N-terminus of the first polypeptide(V_(L), V_(H) or neighboring site thereof), the linker region betweenthe first and second polypeptide regions, or occur in a combination ofthese regions. The design of the carbohydrates site on a proteininvolves examining the structural information known about the proteinand the residues in the proteins involved in antigen binding. Thecarbohydrates sites are chosen to be as far from these residues aspossible so as to prevent disruption of the antigen-binding site. SeeHubbard, S. C., and Ivatt, R. J., Ann. Rev. Biochem. 50:555-583 (1981),for review of synthesis and processing of Asn-linked glycosylation.

The glycosylation sequence may occur adjacent to the (1) nativeC-terminus residue of V_(L) (or V_(H)), (2) the C-terminus of V_(L) (orV_(H)) wherein the C-terminus has a deletion of one or plurality ofamino acid residue(s), such that the remaining N-terminus amino acidresidues of the peptide are sufficient for the glycosylated polypeptideto be capable of binding an antigen, or (3) the C-terminus of V_(L) (orV_(H)) wherein the C-terminus residue has an addition of one orplurality of amino acid residue(s), such that the glycosylatedpolypeptide is capable of binding an antigen. By “native” is intendedthe naturally occurring C-terminus of the immunoglobulin(secondpolypeptide). By “C-terminus,” it is well understood in the artas intending the C-terminus amino acid residue or the C-terminus regionof a polypeptide, which could include up to all of the amino acidresidues of the polypeptide excluding the first N-terminus amino acidresidue of the polypeptide. However, in the present invention,“C-terminus” is intended as the C-terminus amino acid residue of theabove-mentioned three types of C-terminus (1, 2 or 3), unless otherwiseindicated or intended.

Glycosylation sequences were identified and engineered at residueswithin loop sites in regions of the SCA that are diametrically opposedto the antigen binding site. The five loop regions and C-terminalextension chosen as preferred sites of glycosylation are among the mostdistant regions spatially removed from the binding site.

The six furthest portions of an SCA from the antigen binding site are asfollows:

1) The loop made up of residues 11 to 15 in the light chain;

2) The loop made up of residues 77 to 79 in the light chain;

3) The N-terminus of the linker;

4) The loop made up of residues 11 to 15 in the heavy chain;

5) The loop made up of residues 82B, 82C and 83 in the heavy chain; and

6) The C-terminus of the SCA.

The residues are identified as according to Kabat et al., Sequences ofProteins of Immunological Interest, 5th ed., U.S. Dept. Health and HumanServices, Bethesda, Md. (1991). These possible glycosylation sites weredetermined by examining the 4-4-20 mouse Fab structure (see, Whitlow, M.et al., Protein Engng. 8:749-761 (1995), which disclosure isincorporated herein by reference).

After identifying the loops furthest from the antigen binding site, thenucleic and amino acid sequences of each loop are examined for possibleN-linked glycosylation sequences which may be engineered into the loopregion. The best sites are those in which it takes a minimum number ofamino acid changes to generate the Asn-Xaa-Thr/Ser glycosylationsequence. (According to Gavel, Y., and von Heijne, G., Protein Engng.3:433-442 (1990), which disclosure is incorporated herein by reference,Thr occurs in successfully glycosylated tripeptide sequences about twotimes as often as Ser.) This can be performed manually or with acomputer program using sequence homology rules, such as the “GeneWorks”Program from Intelligenetics, Inc. (Mountain View, Calif.). However, theengineered placement of the N of the N-X-S/T sequence anywhere in thesesix identified regions can generate a preferred site for SCAglycosylation.

The design approach described above has been used for the CC49/218 SCA.FIGS. 1A and 1B show the following resulting designs: designedglycosylation sites no. 1 and no. 2 in the light chain of the CC49/218SCA; designed glycosylation site no. 3 in the N-terminal end of thelinker in CC49/218 SCA; designed glycosylation sites no. 4 and no. 5 inthe heavy chain of the CC49/218 SCA; designed glycosylation site no. 6adjacent to the C-terminus of the CC49/218 SCA. Any combination of thesesix sites could be used. The design approach can be used for other SCAs,such as a Kabat consensus V_(k)I/218/V_(H)III, C6.5/218 and A33/218, asshown in FIGS. 6-8, respectively.

The particular nucleotide sequence which is used to introduce anAsn-linked glycosylation sequence into the various positions will dependupon the naturally-occurring nucleotide sequence. The most preferredsites are those in which it takes a minimum number of amino acid changesto generate the Asn-linked glycosylation sequence. For example,glycosylation sequence no. 1 in FIG. 1A was generated by mutating aminoacid 12, Pro (CCT), to Asn (AAC), resulting in an Asn-linkedglycosylation sequence of Asn-Val-Ser. Similarly, other Asn-linkedsequences may be generated. Of course, based on the redundancy of thegenetic code, a particular amino acid may be encoded by multiplenucleotide sequences.

Site-directed mutagenesis is used to change the native protein sequenceto one that incorporates the designed sites of N-linked glycosylation.The mutant protein gene is placed in an expression system that iscapable of glycosylating the protein, such as bacterial cells, yeast orother fungal cells, insect cells or mammalian cells. It may be importantto find a system that uniformly glycosylates the mutant glycoprotein.The mutant glycoprotein can be purified by standard purificationmethods.

Oligonucleotide-directed mutagenesis methods for generating theAsn-linked glycosylation sequences and related techniques formutagenesis of cloned DNA are well known in the art. See, Sambrook etal., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989); Ausubel etal. (eds.),CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons (1987), eachof which disclosure is incorporated herein by reference. A preferredoligonucleotide-directed mutagenesis method for the present invention isaccording to Ho et al., Gene 77:51-59 (1989), which disclosure isincorporated herein by reference. The primer sequences used forgenerating tandem and/or overlapping glycosylation sequences aredisclosed in Example 1, Materials and Methods section, infra.

Synthesis of Multiple Tandem and Overlapping Glycosylation Sequences

A single chain antigen binding molecule (e.g., CC49) with the geneticsequence (polynucleotide) available for glycosylation can be made tocontain the carbohydrate moiety by a post-translation process that isavailable in eukaryotic cells, such as in the yeast Pichia.

The gCC49 was derived from the original clone CC49/218 by incorporatingglycosylation genetic sequences. Several clones were created.

Plasmid pEN270 was transformed into Pichia to give a clone EN279 thatsecretes glycosylated CC49-protein designated as gCC49/2. This clonecontained two glycosylation sequences. The gene product produced by theyeast had over 50% of the total expressed single chain antigen bindingmolecule in the glycosylated form.

Plasmid pEN271 was transformed into Pichia to give a clone EN280 thatsecretes glycosylated CC49 protein designated as gCC49/3. This clonecontained three glycosylation sequences. The gene product produced bythe yeast had over 90% of the single chain antigen-binding protein inthe glycosylated form.

CC49 having exemplary one, two or three glycosylation sequence(s) (inbold) adjacent to the C-terminus are provided below.

C-terminal of CC49 with one glycosylation sequence:

Ser Val Thr Val Ser Asn Lys Thr Ser Stop BamHI (SEQ ID NO:12)

TCA GTC ACC GTC TCC AAC AAG ACC AGT TAA TAG GAT CC (SEQ ID NO:11)

C-terminal of CC49 with two glycosylation sequences:

Ser Val Thr Val Ser Asn Lys Thr Asn Ala Thr Ser Stop BamHI (SEQ IDNO:14)

TCA GTC ACC GTC TCC AAC AAG ACC AAT GCT ACC TCT TAA TAG GATCC (SEQ IDNO:13)

C-terminal of CC49 with three glycosylation sequences:

Ser Val Thr Val Ser Asn Lys Thr Asn Asn Thr Thr Ser Stop BamHI (SEQ IDNO:16)

TCA GTC ACC GTC TCC AAC AAG ACC AAC AAT ACT ACC TCT TAA G GAT CC (SEQ IDNO:15)

CC49 with one glycosylation sequence adjacent to the C-terminus includesthe glycosylation sequence N-K-T (amino acids depicted in bold are thosenecessary for a glycosylation site). CC49 with two tandem glycosylationsequences adjacent to the C-terminus is also shown: N-K-T-N-A-T (SEQ IDNO:17). Additionally, CC49 with three overlapping and tandemglycosylation sequences adjacent to the C-terminus is shown:N-K-T-N-N-T-T (SEQ ID NO:18). Of course, the SCA may have two or more(or at least two), such as three, five, seven, or ten, for example,N-linked glycosylation sequences in tandem, or one or more (or at leastone) set, such as three, five, seven, or ten, for example, sets of twotandem sequences. The SCA may have one or more (or at least one) set,such as two, three, five, seven or ten, for example, sets of twooverlapping sequences.

The glycosylated CC49 was purified by a combination of cation exchangechromatography and anion exchange chromatography. The protein fractionthat contains no carbohydrate was removed by size exclusionchromatography.

Other proteins may also be modified by the addition of tandem oroverlapping glycosylation sequences. Such proteins include, but are notlimited to, disulfide-stabilized Fv fragments (Reiter, Y. et al., NatureBiotech. 14:1239-1245 (1996)), camel immunoglobulins (Muyldermans, S. etal., Protein Engng. 7:1129-1135 (1994)), cancer vaccines, cell adhesionproteins such as selectin, members of the immunoglobulin superfamilyincluding IgG, IgM, IgE, IgD and IgA as well as other immunoglobulinfamily proteins. Included also are therapeutic enzymes such as DNase,RNase, and catabolic enzymes, cytokines, hormones, and growth factorssuch as erythropoietin, GCSF (Granulocyte colony stimulating factor),GMCSF (Granulocyte macrophage colony stimulating factor). Also includedare interleukin-2, alpha-interferon, insulin, human growth hormone, andother blood proteins such as tissue plasminogen activator and FactorVIII and Factor XI. In addition, these methods may be applied tovaccines such as hepatitis B vaccine, AIDS vaccines, lyme diseasevaccines, and other infectious disease vaccines. Use of this technologyalso includes the selective N-linked modification of gene therapyvectors including viral vectors, non-viral vectors, and cellular vectorswhich contain such engineered N-linked glycoproteins.

It is surprising that SCA containing multiple tandem and/or overlappingglycosylation sequences are more completely glycosylated than an SCAcontaining a single glycosylation sequence as carbohydrate attachment atAsn residues near or adjacent to each other would be expected toencounter steric hindrance. Specifically, the two-sequence version wasgreater than 50% modified and the three-sequence version was greaterthan 95% modified compared to the single-site version which was about35-50% modified. Moreover, in the triple sequence version, it isapparent that longer oligosaccharide chains are present. Thus, theinvention relates to (1) a method of producing a polypeptide havingincreased glycosylation, comprising: (a) providing to a polynucleotideencoding the polypeptide at least two tripeptide Asn-linkedglycosylation sequences, wherein each tripeptide glycosylation sequencecomprises Asn-Xaa-Yaa, wherein Xaa is an amino acid other than prolineand Yaa is threonine or serine, and wherein the tripeptide glycosylationsequences are in tandem such that the Asn residues are separated by twoamino acid residues; and (b) expressing the polynucleotide in a hostcell capable of attaching a carbohydrate moiety at the Asn residues, and(2) a polypeptide having increased glycosylation produced by thedescribed process.

The invention further relates to (1) a method of producing a polypeptidehaving increased glycosylation, comprising: (a) providing to apolynucleotide encoding the polypeptide at least one set of twotripeptide Asn-linked glycosylation sequences, wherein each tripeptideglycosylation sequence comprises Asn-Xaa-Yaa, wherein Xaa is an aminoacid other than proline and Yaa is threonine or serine, and wherein thetwo tripeptide glycosylation sequences overlap such that the Asnresidues are adjacent; and (b) expressing the polynucleotide in a hostcell capable of attaching a carbohydrate moiety at the Asn residues, and(2) a polypeptide having increased glycosylation produced by thedescribed process.

The invention also relates to (1) a method of producing a polypeptidehaving increased glycosylation, comprising: (a) providing to apolynucleotide encoding the polypeptide at least two tripeptideAsn-linked glycosylation sequences, wherein each tripeptideglycosylation sequence comprises Asn-Xaa-Yaa, wherein Xaa is an aminoacid other than proline and Yaa is threonine or serine, and wherein thetripeptide glycosylation sequences are in tandem such that the Asnresidues are separated by two amino acid residues; (b) providing to thepolynucleotide at least one set of two tripeptide Asn-linkedglycosylation sequences, wherein the two tripeptide glycosylationsequences overlap such that the Asn residues are adjacent; and (c)expressing the polynucleotide in a host cell capable of attaching acarbohydrate moiety at the Asn residues, and (2) a polypeptide havingincreased glycosylation produced by the described process.

Hosts and Vectors

By “polynucleotide,” is intended DNA, RNA or a genetic sequence. Aftermutating the nucleotide sequence of the SCA, the mutated DNA can beinserted into a cloning vector for further analysis, such as forconfirmation of the DNA sequence. To express the polypeptide encoded bythe mutated DNA sequence, the DNA sequence is operably linked toregulatory sequences controlling transcriptional expression andintroduced into either a prokaryotic or eukaryotic host cell.

Although SCAs are typically produced by prokaryotic host cells,eukaryotic host cells are the preferred host cells. Preferred host cellsinclude plant cells, yeast or other fungal cells, insect cells ormammalian cells. Standard protein purification methods may be used topurify these mutant glycoproteins. Only minor modification to the nativeprotein's purification scheme may be required.

Also provided by the invention are DNA molecules such as purifiedgenetic sequences or plasmids or vectors encoding the SCA of theinvention that have of engineered sequences capable of N-linkedglycosylation. The DNA sequence for the glycosylated SCA polypeptide canbe chosen so as to optimize production in organisms such as plant cells,prokaryotes, yeast or other fungal cells, insect cells or mammaliancells.

The DNA molecule encoding an SCA having Asn-linked glycosylationsequences can be operably linked into an expression vector andintroduced into a host cell to enable the expression of the glycosylatedSCA protein by that cell. A DNA sequence encoding an SCA havingAsn-linked glycosylation sequences may be recombined with vector DNA inaccordance with conventional techniques.

Recombinant hosts as well as methods of using them to produce singlechain proteins of the invention are also provided herein.

The expression of such SCA proteins of the invention can be accomplishedin procaryotic cells. Preferred prokaryotic hosts include, but are notlimited to, bacteria such as Neisseria, Mycobacteria, Streptococci,Chlamydia and E. coli which expresses recombinant heterologous enzymescapable of glycosylation.

Eukaryotic hosts for cloning and expression of such SCA proteins of theinvention include insect cells, yeast, fungi, and mammalian cells (suchas, for example, human or primate cells) either in vivo, or in tissueculture. A preferred host for the invention is Pichia pastoris.

The appropriate DNA molecules, hosts, methods of production, isolationand purification of monovalent, multivalent and fusion forms ofproteins, especially SCA polypeptides, are thoroughly described in theprior art, such as, e.g., U.S. Pat. No. 4,946,778, which disclosure isincorporated herein by reference.

The SCA encoding sequence having Asn-linked glycosylation sequences andan operably linked promoter may be introduced into a recipientprokaryotic or eukaryotic cell either as a non-replicating DNA (or RNA)molecule, which may either be a linear molecule or, more preferably, aclosed covalent circular molecule. Since such molecules are incapable ofautonomous replication, the expression of the desired SCA protein mayoccur through the transient expression of the introduced sequence.Alternatively, permanent expression may occur through the integration ofthe introduced SCA sequence into the host chromosome.

In one embodiment, the SCA sequence can be integrated into the host cellchromosome. Cells which have stably integrated the introduced DNA intotheir chromosomes can be selected by also introducing one or moremarkers which allow for selection of host cells which contain the SCAsequence and marker. The marker may complement an auxotrophy in the host(such as his4, leu2, or ura3, which are common yeast auxotrophicmarkers), biocide resistance, e.g., antibiotics, or resistance to heavymetals, such as copper, or the like. The selectable marker gene caneither be directly linked to the SCA DNA sequence to be expressed, orintroduced into the same cell by co-transfection.

In another embodiment, the introduced sequence will be incorporated intoa plasmid vector capable of autonomous replication in the recipient hostcell. Any of a wide variety of vectors may be employed for this purpose.Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

Any of a series of yeast vector systems can be utilized. Examples ofsuch expression vectors include the yeast 2-micron circle, theexpression plasmids YEP13, YCP and YRP, etc., or their derivatives. Suchplasmids are well known in the art (Botstein et al., Miami Wntr. Symp.19:265-274 (1982); Broach, J. R., In: The Molecular Biology of the YeastSaccharomyces: Life Cycle and Inheritance, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, J. R.,Cell 28:203-204 (1982)).

For a mammalian host, several possible vector systems are available forexpression. One class of vectors utilize DNA elements which provideautonomously replicating extra-chromosomal plasmids, derived from animalviruses such as bovine papilloma virus, polyoma virus, adenovirus, orSV40 virus. A second class of vectors relies upon the integration of thedesired gene sequences into the host chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes may be selected byalso introducing one or more markers which allow selection of host cellswhich contain the expression vector. The marker may provide forprototropy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or resistance to heavy metals, such as copper or the like.The selectable marker gene can either be directly linked to the DNAsequences to be expressed, or introduced into the same cell byco-transformation. Additional elements may also be needed for optimalsynthesis of mRNA. These elements may include splice signals, as well astranscription promoters, enhancers, and termination signals. The cDNAexpression vectors incorporating such elements include those describedby Okayama, H., Mol. Cell. Biol. 3:280 (1983), and others.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH 16a, pNH 18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Preferred vectors for expression inPichia are pHIL-S1 (Invitrogen Corp.) and pPIC9 (Invitrogen Corp.).Other suitable vectors will be readily apparent to the skilled artisan.

Once the vector or DNA sequence containing the constructs has beenprepared for expression, the DNA constructs may be introduced ortransformed into an appropriate host. Various techniques may beemployed, such as transformation, transfection, protoplast fusion,calcium phosphate precipitation, electroporation, or other conventionaltechniques. After the cells have been transformed with the recombinantDNA (or RNA) molecule, the cells are grown in media and screened forappropriate activities. Expression of the sequence results in theproduction of the glycosylated SCA of the present invention.

In the alternative approach, N-linked glycosylation can be achieved invitro by reacting the mutant SCA polypeptides described herein withpurified N-linked glycosylation enzymes and further reacting suchglycosylated SCA with other carbohydrate modifying enzymes.

Poly(alkylene)-Glycol Modification

The straight chain polyalkylene glycols employed in the practice of thepresent invention are of the structural formula

wherein R is selected from the group consisting of hydrogen, loweralkyl, and mixtures thereof, R¹ is selected from the group consisting ofhydrogen and lower alkyl, and n is a positive integer. By “lower alkyl”is meant an alkyl group having from one to four carbon atoms, i.e.,methyl, ethyl, propyl, butyl, and isomers of the foregoing. R ispreferably selected from the group consisting of hydrogen, methyl, andmixtures thereof, R¹ is preferably selected from the group consisting ofhydrogen and methyl, and n is preferably a positive integer of 500 orless. R is most preferably hydrogen, R¹ is most preferably methyl, and nis most preferably an integer of 7 to 150. It will be readily apparentto those skilled in the art that the preferred poly(alkylene glycols)employed in the practice of the present invention are poly(ethyleneglycol), poly(propylene glycol), mixtures thereof, and copolymers ofpoly(ethylene glycol) and poly(propylene glycol), wherein one of theterminal hydroxyl groups of the polymer may be substituted with a loweralkyl group. A preferred polyalkylene glycol for use in the presentinvention is poly(ethylene glycol)-hydrazide.

For convenience, the polyalkylene glycol employed in the practice of thepresent invention will be designated PAG, which term is intended toinclude both compounds wherein R¹ is hydrogen and compounds wherein R¹is alkyl. PEG refers to poly(ethylene glycol) and mPEG refers to methoxypoly(ethylene glycol).

The PAG does not have to be of a particular molecular weight, but it ispreferred that the molecular weight be between about 500 and about40,000; more preferably, between about 2,000 and about 20,000. Thechoice of molecular weight of PAG is made based on the nature of theparticular polypeptide employed, for example, the number of amino orother groups available on the polypeptide for modification. Molecularweights of about 10,000 and about 20,000 are most preferred.

Zalipsky et al., Eur. Pol. J. 19(12):1177-1183 (1983), among others,have described the reaction of methoxy poly(ethylene glycol) withsuccinic anhydride:

It is also known to alkylate mPEG with ethylbromoacetate in the presenceof a base such as K-tertiary butoxide in tertiary butanol,Na-naphthalene in tetrahydrofuran, or butyl lithium in benzene:

The terminal hydroxyl groups of PEG can be transformed into amine,carboxyl, or hexamethyl isocyanate groups. See, for example, Zalipsky etal., 1983, supra. A mixed anhydride derivative of carboxylated mPEG canbe prepared in the presence of triethylamine and then reacted withproteins:

Carboxylated mPEG can also be reacted with hydroxysuccinimide in thepresence of dicyclohexylcarbodiimide and dimethyl formamide for reactionwith protein:

King and Weiner (Int. J. Peptide Protein Res. 16:147 (1980), describethe dithiocarbonate of mPEG:

Beauchamp et al., Analytical Biochem. 131:25-33 (1983), describe theactivation of PEG with 1,1′-carbonyldiimidazole. Reaction of thisderivative with a peptide yields a carbamate linkage:

Veronese et al., Appl. Biochem. & Biotechnol. 11:141-152 (1985),describe the activation of methoxy poly(ethylene glycol) withphenylchloroformates, e.g., 2,4,5-trichlorophenylchloroformate orp-nitrophenylchloroformate. These derivatives are linked to peptides byurethane linkages:

Ueno et al., European Patent Application 87103259.5, form mPEGimidoesters from the corresponding nitriles by reaction with dryhydrogen chloride in the presence of a dehydrated lower alcohol:

Abuchowsk, et al., Cancer Biochem. Biophys. 7.175-186 (1984), havedescribed forming mPEG succinate as described above and then formingmethoxy polyethylene glycolyl succinimidyl succinate (“SS-PEG”) byreaction with hydroxysuccinimide in the presence ofdicyclohexylcarbodiimide:

Sano et al., European Patent Application No. 89107960.0, disclose thephenyl glyoxal derivative of methoxy poly(ethylene glycol), which iscapable of modifying the guanidino groups in peptides:

Zalipsky, in U.S. Pat. No. 5,122,614, describes the activation of PEG byconversion into its N-succinimide carbonate derivative (“SC-PEG”):

Zalipsky et al., J. Macromol. Sci. Chem. A21:839, disclose the aminoacid ester derivative of methoxy poly(ethylene glycol):

Davis et al., U.S. Pat. No. 4,179,337, disclose a hydrazide derivativeof methoxy poly(ethylene glycol), which is capable of modifyingaldehydes and ketones and other functional groups:

It is further disclosed that the bifunctional derivative of PEG, i.e.,polyethylene glycol-bis-succinidyl carbonate (“BSC-PEG”) can be preparedby similar means. The SC-PEG and BSC-PEG compounds are then reacted withamine groups in a protein and attached thereto via urethane (carbamate)linkages.

It will be readily apparent to those skilled in the art that otheractivated PAGs can also be employed in the practice of the presentinvention. The preferred activated PAG for use in the practice of thepresent invention is PEG-hydrazide.

Branched Polymers

The invention further provides for the use of branched, substantiallynon-antigenic polymers for PEGylation of the SCA proteins correspondingto the formula:

(R)_(n)L—A  (II)

wherein (R) includes a water-soluble non-antigenic polymer;

(n)=2 or 3;

(L) is an aliphatic linking moiety covalently linked to each (R); and

(A) represents an activated functional group capable of undergoingnucleophilic substitution. For example, (A) can be a group which iscapable of bonding with biologically active nucleophiles or moietiescapable of doing the same.

In particularly preferred aspects of the invention (R) includes apoly(alkylene oxide) PAO such as poly(ethylene glycol) PEG or mPEG. Itis preferred that each chain have a molecular weight of between about200 and about 12,000 daltons and preferably between about 1,000 andabout 10,000 daltons. Molecular weights of about 5,000 daltons are mostpreferred.

As shown in Formula II, 2 or 3 polymer chains, designated (R) herein,are joined to the aliphatic linking moiety (L). Suitable aliphaticsincluded substituted alkyl diamines and triamines, lysine esters andmalonic ester derivatives. The linking moieties are preferablynon-planar, so that the polymer chains are not rigidly fixed. Thelinking moiety (L) is also a means for attaching the multiple polymerchains or “branches” to (A), the moiety through which the polymerattaches to the SCA protein.

(L) preferably includes a multiply-functionalized alkyl group containingup to 18, and more preferably between 1-10 carbon atoms. A heteroatomsuch as nitrogen, oxygen or sulfur may be included within the alkylchain. The alkyl chain may also be branched at a carbon or nitrogenatom. In another aspect of the invention, (L) is a single nitrogen atom.

(L) and (R) are preferably joined by a reaction between nucleophilicfunctional groups on both (R) and (L). Each (R) is suitablyfunctionalized to undergo nucleophilic substitution and bond with (L).Such functionalization of polymers is readily apparent to those ofordinary skill in the art.

A wide variety of linkages are contemplated between (R) and (L).Urethane (carbamate) linkages are preferred. The bond can be formed, forexample, by reacting an amino group such as 1,3-diamino-2-propanol withmethoxypolyethylene glycol succinimidyl carbonate as described in U.S.Pat. No. No. 5,122,614. Amide linkages, which can be formed by reactingan amino-terminated non-antigenic polymer such as methoxypolyethyleneglycol-amine (mPEG amine) with an acyl chloride functional group.Examples of other such linkages include ether, amine, urea, and thio andthiol analogs thereof, as well as the thio and thiol analogs of theurethane and amide linkages discussed supra.

The moiety (A) of Formula II represents groups that “activate” thebranched polymers of the present invention for conjugation withbiologically active materials. (A) can be a moiety selected from:

1. Functional groups capable of reacting with an amino group such as:

a) carbonates such as the p-nitrophenyl or succinimidyl;

b) carbonyl imidazole;

c) azlactones;

d) cyclic imide thiones; or

e) isocyanates or isothiocyanates.

2. Functional groups capable of reacting with carboxylic acid groups andreactive with carbonyl groups such as:

a) primary amines; or

b) hydrazine and hydrazide functional groups such as the acylhydrazides, carbazates, semicarbamates, thiocarbazates, etc.

3. Functional groups capable of reacting with mercapto or sulfhydrylgroups such as phenyl glyoxals; see, for example, U.S. Pat. No.5,093,531.

4. Other nucleophiles capable of reacting with an electrophilic center.A non-limiting list includes, for example, hydroxyl, amino, carboxyl,thiol groups, active methylene and the like.

The moiety (A) can also include a spacer moiety located proximal to thealiphatic linking moiety (L). The spacer moiety may be a heteroalkyl,alkoxyl, alkyl containing up to 18 carbon atoms or even an additionalpolymer chain. The spacer moieties can be added using standard synthesistechniques.

The branched polymers, generally, U-PAO's or U-PEG's, are formed usingconventional reaction techniques known to those of ordinary skill in theart.

These umbrella-like branched polymers of the present invention (U-PAO'sor U-PEG's) react with biologically active nucleophiles to formconjugates. The point of polymer attachment depends upon the functionalgroup (A). For example, (A) can be a succinimidyl succinate or carbonateand react with ε-amino lysines. The branched polymers can also beactivated to link with any primary or secondary amino group, mercaptogroup, carboxylic acid group, reactive carbonyl group or the like foundon biologically active polypeptides. Other groups are apparent to thoseof ordinary skill in the art.

One of the main advantages of the use of the branched polymers is thatthe branching imparts an umbrella-like three dimensional protectivecovering to the materials they are conjugated with. This contrasts withthe string-like structure of the straight chain polymers discussed,supra. An additional advantage of the branched polymers is that theyprovide the benefits associated with attaching several strands ofpolymers to a SCA protein or carbohydrate moiety but requiresubstantially fewer conjugation sites. The desired properties ofPEGylation are realized and the loss of bioactivity is minimized.

One or more of the activated branched polymers can be attached to abiologically active nucleophile, such as an SCA protein, by standardchemical reactions. The conjugate is represented by the formula:

[(R)_(n)L′—A¹]_(z)−(nucleophile)  (III)

wherein (R) is a water-soluble substantially non-antigenic polymer; n=2or 3; (L) is an aliphatic linking moiety; (A¹) represents a linkagebetween (L) and the nucleophile and (z) is an integer≧1 representing thenumber of polymers conjugated to the biologically active nucleophile.The upper limit for (z) will be determined by the number of availablenucleophilic attachment sites and the degree of polymer attachmentsought by the artisan. The degree of conjugation can be modified byvarying the reaction stoichiometry using well-known techniques. Morethan one polymer conjugated to the nucleophile can be obtained byreacting a stoichiometric excess of the activated polymer with thenucleophile.

Activated PAO can be attached to the carbohydrate moiety using themethod generally described in Sea et al., Immunoconjugates, Vogel, C.Ed., Oxford University Press, p. 189 (1987), which disclosure isincorporated herein by reference. Briefly, the glycosylated SCA isoxidized with sodium periodate which provides an aldehyde group to whichthe PAO can bind. This reaction is stabilized by sodium borohydride. PAOattachment to polypeptides or glycopolypeptides is also described, forexample, in Zalipsky, S, et al., WO 92/16555, which disclosure isincorporated herein by reference.

Conjugates

Upon production of the glycosylated SCA of the present invention, theglycosylated SCA may further be modified by conjugating a diagnostic ortherapeutic agent to the carbohydrate moiety of the SCA. The generalmethod of preparing an antibody conjugate according to the invention isdescribed in Shih, L. B., et al., Cancer Res. 51:4192 (1991); Shih, L.B., and D. M. Goldenberg, Cancer Immunol. Immunother. 31:197 (1990);Shih, L. B., et al., Intl. J. Cancer 46:1101 (1990); Shih, L. B., etal., Intl. J. Cancer 41:832 (1988), which disclosures are allincorporated herein by reference. The indirect method involves reactingan antibody (or SCalif.), whose carbohydrate portion has been oxidized,with a carrier polymer loaded with one or plurality of peptide, lipid,nucleic acid, drug, toxin, chelator, boron addend or detectable labelmolecule(s).

Alternatively, the glycosylated SCA may be directly conjugated with adiagnostic or therapeutic agent. The general procedure is analogous tothe indirect method of conjugation except that a diagnostic ortherapeutic agent is directly attached to an oxidized sFv component. SeeHansen et al., U.S. Pat. No. No. 5,443,953, which disclosure isincorporated herein by reference.

The glycosylated SCA can be attached to a derivative of the particulardrug, toxin, chelator, boron addend or label to be loaded, in anactivated form, preferably a carboxyl-activated derivative, prepared byconventional means, e.g., using dicyclohexylcarbodiimide (DCC) or awater soluble variant thereof, to form an intermediate adduct.

Many drugs and toxins are known which have a cytotoxic effect on tumorcells or microorganisms that may infect a human and cause a lesion, inaddition to the specific illustrations given above. They are to be foundin compendia of drugs and toxins, such as the Merck Index and the like.Any such drug can be loaded onto a carrier or directly onto acarbohydrate moiety of SCA by conventional means well known in the art,and illustrated by analogy to those described above.

Chelators for radiometals or magnetic resonance enhancers are also wellknown in the art. Typical are derivatives of ethylenediaminetetraaceticacid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). Thesetypically have groups on the side chain by which the chelator can beattached to a carrier or directly onto a carbohydrate moiety of SCA.Such groups include, e.g., a benzylisothiocyanate, by which the DTPA orEDTA can be coupled to the reactive group of an SCA.

Labels such as radioisotopes, enzymes, fluorescent compounds, electrontransfer agents, and the like can also be linked to carrier or directlyonto a carbohydrate moiety of SCA by conventional methods well known tothe art. These labels and the SCA conjugates prepared from them can beused for immunoassays and for immunohistology, much as the SCA conjugateprepared by direct attachment of the labels to the SCA. However, theloading of the conjugates according to the present invention with aplurality of labels can increase the sensitivity of assays orhistological procedures, where only low extent of binding of the SCA totarget antigen is achieved.

Boron addends, e.g., carboranes, when attached to single-chain antigenbinding molecules and targeted to lesions, can be activated by thermalneutron irradiation and converted to radioactive atoms which decay byalpha emission to produce highly cytotoxic short-range effects. Highloading of boron addends, as well as of magnetic resonance enhancingions, is of great importance in potentiating their effects. Carboranescan be made with carboxyl functions on pendant side chains, as is wellknown in the art.

Loading of drugs on the carrier will depend upon the potency of thedrug, the efficiency of SCA targeting and the efficacy of the conjugateonce it reaches its target. In most cases, it is desirable to load atleast 20, preferably 50, and often 100 or more molecules of a drug on acarrier. The ability to partially or completely detoxify a drug as aconjugate according to the invention, while it is in circulation, canreduce systemic side effects of the drug and permit its use whensystemic administration of the unconjugated drug would be unacceptable.Administration of more molecules of the drug, but conjugated to the SCAon a carrier, according to the present invention, permits therapy whilemitigating systemic toxicity.

Toxins will often be less heavily loaded than drugs, but it will stillbe advantageous to load at least 5, preferably 10 and in some cases 20or more molecules of toxin on a carrier and load at least one carrierchain on the SCA for targeted delivery.

The above-described conjugation of a diagnostic or therapeutic agent isalso intended with glycosylated SCA further conjugated to polyalkyleneoxide, at the carbohydrate and/or polymer moiety. Conjugation ofpoly(ethylene glycol) or poly(alkylene oxide) with small organicmolecules is described in Greenwald, R. B., Exp. Opin. Ther. Patents7:601-609 (1997), Enzon Inc., WO 95/11020, and Enzon Inc., WO 96/23794,which disclosures are all incorporated herein by reference. Compositionsbased on the use of various linker groups between the PEG ballast andthe active drug are described in WO 96/23794.

Uses

One of the major utilities of the glycosylated SCA is itsbifunctionality (or multifunctionality, including tri-, quadri-, etc.),in which one specificity is for one type of hapten or antigen, and thesecond specificity is for a second molecule or receptor. A glycosylatedSCA molecule having two distinct binding specificities has manypotential uses. For instance, the carbohydrate moiety may be specificfor a cell-surface epitope of a target cell, such as a tumor cell orother undesirable cell. The antigen-binding site may be specific for acell-surface epitope of an effector cell, such as the CD3 protein of acytotoxic T-cell. In this way, the glycosylated SCA protein may guide acytotoxic cell to a particular class of cells that are to bepreferentially attacked. Alternatively, both targets, the antigen andthe carbohydrate receptor can be on the same cell such that one targetmodulates binding specificity and the other target influences uptake orinternalization.

Mannose-specific lectins are reported to be produced on the surfacefimbria of enterobacterial species such as E. coli, Salmonella, andPseudomonas. Such bacteria might be bound (extensively) to theoligosaccharides of glyco-SCA, while the SCA specificity is directed toan immune cell or otherwise promotes the microbe's clearance. Similarly,mannose specific receptors on tumor cells could have similarapplication. The bacterial lectins are also thought to be important incell adhesion to host and infection suggesting another application.

Carbohydrate moieties on cell, viral or particle surfaces are majordeterminants of their identity. Using the SCA specificity to bind to thecell, viral or particle surfaces, and having the oligosaccharide moietyproject out may give that entity a new identity for interaction withother cells, virus and proteins.

A diagnostic or therapeutic agent is a molecule or atom which isconjugated to an antibody and useful for diagnosis and for therapy. Theimmunoreactivity of the antibody is retained. Diagnostic or therapeuticagents include drugs, toxins, chelators, boron compounds and detectablelabels. See “Conjugates” section, supra, for further details.

The diagnostic or therapeutic agent may be, but is not limited to, atleast one selected from a nucleic acid, a compound, a protein, anelement, a lipid, an antibody, a saccharide, an isotope, a carbohydrate,an imaging agent, a lipoprotein, a glycoprotein, an enzyme, a detectableprobe, and antibody or fragment thereof, or any combination thereof,which may be detectably labeled as for labeling antibodies, as describedherein. Such labels include, but are not limited to, enzymatic labels,radioisotope or radioactive compounds or elements, fluorescent compoundsor metals, chemiluminescent compounds and bioluminescent compounds.Alternatively, any other known diagnostic or therapeutic agent can beused in a method of the present invention.

A therapeutic agent used in the present invention may have a therapeuticeffect on the target cell, the effect selected from, but not limited to,correcting a defective gene or protein, a drug action, a toxic effect, agrowth stimulating effect, a growth inhibiting effect, a metaboliceffect, a catabolic affect, an anabolic effect, an antiviral effect, anantibacterial effect, a hormonal effect, a neurohumoral effect, a celldifferentiation stimulatory effect, a cell differentiation inhibitoryeffect, a neuromodulatory effect, an antineoplastic effect, ananti-tumor effect, an insulin stimulating or inhibiting effect, a bonemarrow stimulating effect, a pluripotent stem cell stimulating effect,an immune system stimulating effect, and any other known therapeuticeffects that may be provided by a therapeutic agent delivered to a cellvia a delivery system according to the present invention.

The SCA conjugate may be used for protection, suppression or treatmentof infection or disease. By the term “protection” from infection ordisease as used herein is intended “prevention,” “suppression” or“treatment.” “Prevention” involves administration of a glycosylated SCAconjugate prior to the induction of the disease. “Suppression” involvesadministration of the composition prior to the clinical appearance ofthe disease.

“Treatment” involves administration of the protective composition afterthe appearance of the disease. It will be understood that in human andveterinary medicine, it is not always possible to distinguish between“preventing” and “suppressing” since the ultimate inductive event orevents may be unknown, latent, or the patient is not ascertained untilwell after the occurrence of the event or events. Therefore, it iscommon to use the term “prophylaxis” as distinct from “treatment” toencompass both “preventing” and “suppressing” as defined herein. Theterm “protection,” as used herein, is meant to include “prophylaxis.”

Such additional therapeutic agents which can further comprise atherapeutic agent or composition of the present invention may beselected from, but are not limited to, known and new compounds andcompositions including antibiotics, steroids, cytotoxic agents,vasoactive drugs, antibodies and other therapeutic modalities.Non-limiting examples of such agents include antibiotics used in thetreatment of bacterial shock, such as gentamycin, tobrarnycin,nafcillin, parenteral cephalosporins, etc; adrenal corticosteroids andanalogs thereof, such as methyl prednisolone, mitigate the cellularinjury caused by endotoxins; vasoactive drugs, such as alpha receptorblocking agent (e.g., phenoxybenzamine), beta receptor agonists (e.g.,isoproterenol), and dopamine are agents suitable for treating septicshock.

Glycosylated SCA of the invention may also be used for diagnosis ofdisease and to monitor therapeutic response. Other uses of glycosylatedSCA proteins are specific targeting of pro-drug activating enzymes totumor cells by a bispecific molecule with specificity for tumor cellsand enzyme. Glycosylated SCA may be used for specific delivery of drugto an in vivo target, such as a tumor, delivery of radioactive metalsfor tumor radioimmunodiagnosis or radioimmunotherapy (Goldenberg, D. M.,Am. J. Med. 94:297 (1993)), nonradioactive metals in applications suchas with boron/uranium-neutron capture therapy (Ranadive, G. N., et al.,Nucl. Med. Biol. 20:1 (1993); Barth, R. F., et al., Bioconjug. Chem.5:58 (1994)), and nuclear magnetic resonance imaging (Sieving, P. F., etal., Bioconjug. Chem. 1:65 (1990)). This list is illustrative only, andany use for which an oligosaccharide-specificity is appropriate comeswithin the scope of this invention.

The invention also extends to uses for the glycosylated SCA proteins inpurification and biosensors. Affinity purification is made possible byaffixing the glycosylated SCA protein to a support, with theantigen-binding sites exposed to and in contact with the ligand moleculeto be separated, and thus purified. Biosensors generate a detectablesignal upon binding of a specific antigen to an antigen-bindingmolecule, with subsequent processing of the signal. Glycosylated SCAproteins, when used as the antigen-binding molecule in biosensors, maychange conformation upon binding, thus generating a signal that may bedetected.

The invention is also directed to a method of detecting an antigensuspected of being in a sample by contacting the sample with theglycosylated SCA that is labeled by its carbohydrate moiety. A samplemay comprise at least one compound, mixture, surface, solution,emulsion, suspension, mixture, cell culture, fermentation culture, cell,tissue, secretion and/or derivative or extract thereof.

Such samples can also include, e.g., animal tissues, such as blood,lymph, cerebrospinal fluid (CNS), bone marrow, gastrointestinalcontents, and portions, cells or internal and external secretions ofskin, heart, lung and respiratory system, liver, spleen, kidney,pancreas, gall bladder, gastrointestinal tract, smooth, skeletal orcardiac muscle, circulatory system, reproductive organs, auditorysystem, the autonomic and central nervous system, and extracts or cellcultures thereof. Such samples can be measured using methods of thepresent invention in vitro, in vivo and in situ.

Such samples can also include environmental samples such as earth, airor water samples, as well as industrial or commercial samples such ascompounds, mixtures, surfaces, aqueous chemical solutions, emulsions,suspensions or mixtures.

Additionally, samples that can be used in methods of the presentinvention include cell culture and fermentation media used for growth ofprokaryotic or eukaryotic cells and/or tissues, such as bacteria, yeast,mammalian cells, plant cells and insect cells.

Essentially all of the uses for which monoclonal or polyclonalantibodies, or fragments thereof, have been envisioned by the prior art,can be addressed by the glycosylated SCA proteins of the presentinvention. These uses include detectably-labeled forms of theglycosylated SCA protein. Types of labels are well-known to those ofordinary skill in the art. They include radiolabeling, chemiluminescentlabeling, fluorochromic labeling, and chromophoric labeling. Other usesinclude imaging the internal structure of an animal (including a human)by administering an effective amount of a labeled form of theglycosylated SCA protein and measuring detectable radiation associatedwith the animal. They also include improved immunoassays, includingsandwich immunoassay, competitive immunoassay, and other immunoassayswherein the labeled antibody can be replaced by the glycosylated SCAprotein of this invention. See, e.g., Kohler et al., Nature 256:495(1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al.,Eur. J. Immunol. 6:292 (1976); Hammerling et al., In: MonoclonalAntibodies and T-Cell Hybridomas, pp. 563-681, Elsevier, N (1981);Sambrook et al., Molecular Cloning—A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory (1989).

The above described uses are also intended with glycosylated SCAconjugated to polyalkylene oxide, especially for, for example, reducedimmunogenicity and antigenicity and longer lifetimes in the bloodstream.

Administration

Administration of glycosylated SCA conjugates of the invention for invivo diagnostic and therapeutic applications will be by analogousmethods to conjugates of the same or similar drugs, toxins, chelators,boron adducts or detectable labels where the diagnostic or therapeuticprinciple is directly linked to the antibody or a loaded carrier islinked by random binding to amine or carboxyl groups on amino acidresidues of the antibody in a non-site-specific manner.

Conjugates of the present invention (immunoconjugates) can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, such as by admixture with a pharmaceutically acceptablecarrier vehicle. Suitable vehicles and their formulation are described,for example, in Remington's Pharmaceutical Sciences, 18th ed., Osol, A.,ed., Mack, Easton Pa. (1990). In order to form a pharmaceuticallyacceptable composition suitable for effective administration, suchcompositions will contain a therapeutically effective amount of theimmunoconjugate, either alone, or with a suitable amount of carriervehicle.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to complex or absorb the immunoconjugate of thepresent invention. The controlled delivery may be exercised by selectingappropriate macromolecules (for example, polyesters, polyamino acids,polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate). The rate of drug releasemay also be controlled by altering the concentration of suchmacromolecules. Another possible method for controlling the duration ofaction comprises incorporating the therapeutic agents into particles ofa polymeric substance such as polyesters, polyamino acids, hydrogels,poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, itis possible to entrap the immunoconjugate of the invention inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, by the use ofhydroxymethylcellulose or gelatin-microcapsules orpoly(methylmethacrylate) microcapsules, respectively, or in a colloiddrug delivery system, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, nanocapsules, or in macroemulsions. Suchteachings are disclosed in Remington's Pharmaceutical Sciences, 16thed., Osol, A., ed., Mack, Easton Pa. (1990).

The immunoconjugate may be provided to a patient by means well known inthe art. Such means of introduction include oral means, intranasalmeans, subcutaneous means, intramuscular means, intravenous means,intra-arterial means, or parenteral means. Intravenous, intraarterial orintrapleural administration is normally used for lung, breast, andleukemic tumors. Intraperitoneal administration is advised for ovariantumors. Intrathecal administration is advised for brain tumors andleukemia. Subcutaneous administration is advised for Hodgkin's disease,lymphoma and breast carcinoma. Catheter perfusion is useful formetastatic lung, breast or germ cell carcinomas of the liver.Intralesional administration is useful for lung and breast lesions.

For therapeutic or diagnostic applications, compositions according tothe invention may be administered parenterally in combination withconventional injectable liquid carriers such as sterile pyrogen-freewater, sterile peroxide-free ethyl oleate, dehydrated alcohol, orpropylene glycol. Conventional pharmaceutical adjuvants for injectionsolution such as stabilizing agent, solubilizing agents and buffers,such as ethanol, complex forming agents such as ethylene diaminetetraacetic acid, tartrate and citrate buffers, and high-molecularweight polymers such as polyethylene oxide for viscosity regulation maybe added. Such compositions may be injected intramuscularly,intraperitoneally, or intravenously.

Further non-limiting examples of carriers and diluents include albuminand/or other plasma protein components such as low density lipoproteins,high density lipoproteins and the lipids with which these serum proteinsare associated. These lipids include phosphatidyl choline, phosphatidylserine, phosphatidyl ethanolamine and neutral lipids such astriglycerides. Lipid carriers also include, without limitation,tocopherol.

At least one glycosylated SCA linked to a therapeutic agent according tothe invention may be administered by any means that achieve theirintended purpose, for example, to treat various pathologies, such ascell inflammatory, allergy, tissue damage or other related pathologies.

A typical regimen for preventing, suppressing, or treating variouspathologies comprises administration of an effective amount of an SCAconjugate, administered over a period of one or several days, up to andincluding between one week and about 24 months.

It is understood that the dosage of the present invention administeredin vivo or in vitro will be dependent upon the age, sex, health, andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired. The ranges ofeffective doses provided below are not intended to limit the inventionand represent preferred dose ranges. However, the most preferred dosagewill be tailored to the individual subject, as is understood anddeterminable by one of skill in the art, without undue experimentation.See, e.g., Berkow et al., eds., Merck Manual, 16th edition, Merck andCo., Rahway, N.J. (1992); Goodman et al., eds., Goodman and Gilman's ThePharmacological Basis of Therapeutics, 8th edition, Pergamon Press,Inc., Elmsford, N.Y. (1990); Avery's Drug Treatment: Principles andPractice of Clinical Pharmacology and Therapeutics, 3rd edition, ADISPress, LTD., Williams and Wilkins, Baltimore, Md. (1987), Ebadi,Pharmacology, Little, Brown and Co., Boston (1985), Katzung, Basic andClinical Phamacology, Appleton and Lange, Norwalk, Conn. (1992), whichreferences and references cited therein, are entirely incorporatedherein by reference.

The total dose required for each treatment may be administered bymultiple doses or in a single dose. Effective amounts of adiagnostic/pharmaceutical compound or composition of the presentinvention are from about 0.001 μg to about 100 mg/kg body weight,administered at intervals of 4-72 hours, for a period of 2 hours to 5years, or any range or value therein, such as 0.01-1.0, 1.0-10, 10-50and 50-100 mg/kg, at intervals of 1-4, 6-12, 12-24 and 24-72 hours, fora period of 0.5, 1.0-2.0, 2.0-4.0 and 4.0-7.0 days, or 1, 1-2, 2-4, 4-52or more weeks, or 1, 2, 3-10, 10-20, 20-60 or more years, or any rangeor value therein.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions, which may containauxiliary agents or excipients which are known in the art.Pharmaceutical compositions such as tablets and capsules can also beprepared according to routine methods. See, e.g., Berker, supra,Goodman, supra, Avery, supra and Ebadi, supra, which disclosures areentirely incorporated herein by reference, including all referencescited therein.

Pharmaceutical compositions comprising at least one type of SCAconjugate of the invention, or, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 types ofSCA conjugates, of the present invention may be contained in an amounteffective to achieve its intended purpose. In addition to at least oneSCA conjugate, a pharmaceutical composition may contain suitablepharmaceutically acceptable carriers, such as excipients, carriersand/or auxiliaries which facilitate processing of the active compoundsinto preparations which can be used pharmaceutically.

Pharmaceutical compositions may also include suitable solutions foradministration intravenously, subcutaneously, dermally, orally,mucosally or rectally, and contain from about 0.01 to 99 percent,preferably from about 20 to 75 percent of active component (i.e., theSCA) together with the excipient. Pharmaceutical compositions for oraladministration include tablets and capsules. Compositions which can beadministered rectally include suppositories. See, e.g., Berker, supra,Goodman, supra, Avery, supra and Ebadi, supra. Additional lipid andlipoprotein drug delivery systems that may be included herein aredescribed more fully in Annals N.Y. Acad. Sci. 507:775-88, 98-103, and252-271, which disclosure is incorporated herein by reference.

The compositions may also be formulated into orally administrablecompositions containing one or more physiologically compatible carriersor excipients, and may be solid or liquid in form. These compositionsmay, if desired, contain conventional ingredients such as bindingagents, for example, syrups, acacia, gelatin, sorbitol, tragacanth, orpolyvinylpyrrolidone; fillers, such as lactose, mannitol, starch,calcium phosphate, sorbitol, cyclodextran, or methylcellulose;lubricants such as magnesium stearate, high molecular weight polymerssuch as polyethylene glycols, high molecular weight fatty acids such asstearic acid or silica; disintegrants such as starch; acceptable wettingagents as, for example, sodium lauryl sulfate.

The oral compositions may assume any convenient form, such as tablets,capsules, lozenges, aqueous or oily suspensions, emulsions, or dryproducts suitable for reconstitution with water or other liquid mediumprior to use. The liquid oral forms may, of course, contain flavors,sweeteners, preservatives such as methyl or propyl p-hydroxybenzoates;suspending agents such as sorbitol, glucose or other sugar syrup,methyl, hydroxymethyl, or carboxymethyl celluloses or gelatin;emulsifying agents such as lecithin or sorbitan monooleate or thickeningagents. Non-aqueous compositions may also be formulated which compriseedible oils as, for example, fish-liver or vegetable oils. These liquidcompositions may conveniently be encapsulated in, for example, gelatincapsules in a unit dosage amount.

The pharmaceutical compositions according to the present invention mayalso be administered, if appropriate, either topically as an aerosol or,formulated with conventional bases as a cream or ointment.

The pharmaceutical compositions of the present invention can also beadministered by incorporating the active ingredient into colloidalcarriers, such as liposomes. Liposome technology is well known in theart, having been described by Allison et al., Nature 252:252-254 (1974),and Dancy et al., J. Immunol. 120:1109-1113 (1978).

The above described administration of the compositions also include theglycosylated SCA conjugated to polyalkylene oxide.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Synthesis of Asn-linked Glycosylation Sequences inCC49/218 and Expression of the Glycosylated SCA

The CC49 monoclonal antibody was developed by Dr. Jeffrey Schlom's groupat Laboratory of Tumor Immunology and Biology, National CancerInstitute. It binds specifically to the pan-carcinoma tumor antigenTAG-72 (see, Muraro, R., et al., Cancer Res. 48:4588-4596 (1988)). TheSCA gene version of CC49 has been described by Milenic et al., CancerRes. 51:6363-6371 (1991). Oligonucleotide-directed mutagenesis wasemployed to create Asn-linked glycosylation sequences in CC49/218 (the218 linker is described in the “Detailed Description of the PreferredEmbodiments” section, supra), as shown in the DNA sequences presented inFIGS. 1A and 1B (i.e., (1) two V_(L) changes; (2) two V_(H) changes; (3)one linker change; (4) one C-terminal change). Oligonucleotide-directedmutagenesis was also employed to create two or three tandem oroverlapping glycosylation sites in CC49/218. Additionally, mutant geneswere made having all six changes, five changes excluding C-terminuschange, and C-terminus plus linker changes. These mutant CC49 genes andthe nonmutated CC49 SCA gene were individually ligated into the Pichiatransfer plasmid pHIL-S1 (Invitrogen Corp.) and transformed into theyeast Pichia pastoris. Detailed protocols for these procedures arepresented in the Pichia Expression Kit Instruction Manual Cat. No.X1710-01 (1994) from Invitrogen Corporation. The CC49 gene variants wereplaced behind a yeast signal sequence in these constructions and theintegrated SCA genes in the yeast transformants were tested forsecretion of the SCA protein or glycoprotein products. Evaluation ofexpression was done by Coomassie Blue staining of SDS-PAGE gels.

The unmodified CC49/218 SCA (˜27 kDa) was expressed (secreted) at highlevels in recombinant Pichia (about 20-100 mg/l depending on integratedgene copy number). All of the mutant genes described above gavedetectable (though reduced) expression of secreted glyco-SCA plusunglycosylated SCA as observed by protein bands in the ˜27-35 kDa range.Each glycosylation event would be predicted to add ˜2-3 kDa of masssince Pichia is reported to add oligomannose chains of about 8-14residues to the core N-Acetylglucosamines (Cregg, J. M., et al.,Bio/Technol. 11:905-910 (1993). The C-terminal mutant gave the highestlevel of expression (secretion) and two prominent bands of ˜27 kDa and˜30 kDa in about equal proportions. Expression of secreted glyco-SCA bythe mutants is summarized in Table 2.

TABLE 2 Glyco- CC49 SCA Clone SCA Unmodified SCA CC49 parent (EN225) −++++ Linker mutant + ++ C-terminal mutant ++ ++ (EN235) C-terminus pluslinker ++ ++ mutant (EN236) Five changes mutant + + All six changesmutant + +

These results indicate that there were some glycosylation of all mutantSCA proteins and show that the C-terminal mutant gave the highestexpression (secretion) of glyco-SCA. This mutant was chosen for moredetailed study.

The purified CC49/218 SCA from E. coli GX9251 and the unpurified culturesupernatant from P. pastoris EN225 were assayed for direct binding toantigen, bovine submaxillary mucin, by ELISA. As shown in FIGS. 2A and2B, the parent CC49/218 SCA product from both E. coli and Pichia wereshown to be active in binding bovine submaxillary mucinby ELISA. Thisindicates that CC49/218 SCA produced in Pichia is active.

The C-terminal plus linker double mutant (EN236) CC49/218 SCA was run ona SDS-PAGE gel (FIG. 3). The upper band of the doublet was selectivelystained by using the GlycoTrack Carbohydrate Kit K-050 from OxfordGlycoSystems as described by the manufacturer. The lower ˜27 kDa(unmodified) band was unstained indicating that the ˜30 kDa band was aglycoprotein.

Further, the C-terminus mutant (EN235) and C-terminal plus linker doublemutant (EN236) CC49/218 SCAs were digested with the glycosidasePeptide-N-glycosidase (PNGase) F or Endo-glycosidase H (OxfordGlycosystems) which will specifically cleave Asparagine-linked(N-linked) carbohydrate from the polypeptide chain. Following PNGase For Endo-glycosidase H treatment, the samples were analyzed by SDS-PAGEwhich showed that the former doublets (˜27 and ˜30 kDa) were convertedto a single ˜27 kDa band by Coomassie staining. As shown in FIG. 3,Western analysis using an anti-CC49 SCA rabbit serum antibody (HRP Inc.)confirmed that both bands of the expressed protein doublet from theC-terminus and C-terminal plus linker double mutants react with thisCC49 specific antibody.

The C-terminus mutant CC49/218 SCA doublet proteins were bound to abovine submaxillary mucin-Sepharose affinity column and eluted byincreasing urea concentrations. As shown in FIGS. 4A and 4B, the boundand eluted doublet appeared in equal stoichiometry as in the startingsample indicating that the glyco-SCA maintains mucin-bindingspecificity.

CC49/218 SCA from EN235 culture supernatant was incubated with molarexcess of Con A Sepharose resin (Pharmacia Biotech). The unboundsupernatant fraction was removed, and the bound fraction was eluted withalpha-D-methylmannoside. As shown in FIGS. 5A and 5B(a-c), theglycosylated bound fraction was ˜30 kDa whereas the unglycosylatedunbound fraction was ˜27 kDa.

CC49 SCA with one, two or three glycosylation sequences in theC-terminus are shown in FIG. 9. Western blot analysis of CC49 SCA withone glycosylation site (FIG. 9, lanes 3 and 4) adjacent to theC-terminus shows that it is a mixture of modified (˜30 kDa band) andunmodified (˜27 kDa band) polypeptides. CC49 SCA with two glycosylationsites (FIG. 9, lanes 5 and 6) had a smaller percentage of unmodifiedpolypeptides, a mixture of one and two glycosylation site species, and ahyperglycosylated species (˜43 kDa band). CC49 with three glycosylationsites (FIG. 9, lanes 7 and 8) had virtually no unmodified polypeptides,a mixture of two glycosylation site species and the hyperglycosylatedspecies.

The SCA containing three glycosylation sequences (FIG. 9, lanes 7 and 8)had mainly two attached oligosaccharides or hyperglycosylated species(and virtually no unmodified protein). The higher molecular weighthyperglycosylated species may include both the three positionsattachments and/or more extensive longer-chain oligosaccharideattachments at one or more positions of glycosylation.

The unmodified CC49 SCA (E. coli CC49 and P. pastoris EN225), EN236 (oneC-terminal glycosylation sequence), EN279 (two glycosylation sequences),and EN280 (three glycosylation sequences) were assayed for directbinding to antigen, bovine submaxillary mucin, by ELISA (FIG. 10). Thetwo controls, BSA and GS115 (P. pastoris host) showed little mucinbinding activity.

Materials and Methods

Materials. The gene for CC49/218 SCA was obtained from plasmid pGX5608(Enzon, Inc.). The complete DNA sequence of CC49/218 SCA has beenreported (Filpula, D., et al., “Production of Single-chain Fv Monomersand Multimers, In: Antibody Engineering: A Practical Approach (J.McCafferty, H. Hoogenboom, and D. J. Chiswell, eds., Oxford UniversityPress, Oxford, UK), pp. 253-268 (1996)). Oligonucleotides weresynthesized using a Millipore Cyclone DNA Synthesizer. The GlycoTrackCarbohydrate Detection Kit K-050, Endoglycosidase H, andPeptide-N-Glycosidase F were purchased from Oxford GlycoSystems(Rosedale, N.Y.). Pre-cast polyacrylamide slab gels (4-20%) wereobtained from Novex Corporation (San Diego, Calif.). Bovine submaxillarymucin type I, porcine submaxillary mucin type III and CNBr-activatedSepharose 4B were purchased from Sigma Inc. (St. Louis, Mo.). Con ASepharose was obtained from Pharmacia Biotech (Piscataway, N.J.).Purified CC49/218 SCA protein derived from E. coli GX9251 was obtainedfrom Enzon, Inc. Rabbit anti-CC49/218 SCA polyclonal antibody wasobtained from HRP Inc. (Denver, Pa.). Mouse anti-CC49/218 polyclonalantibody was obtained from Enzon, Inc.

SCA gene constructions. The CC49/218 SCA gene from plasmid pGX5608 wasmodified by oligonucleotide-directed mutagenesis using the procedure ofHo et al., Gene 77:51-59 (1989). The six designated changes for N-linkedglycosylation (N-X-T/S) are indicated in FIGS. 1A and 1B. DNA sequenceanalysis using T7 Sequenase version 2.0 (Amersham Corporation, ArlingtonHeights, Ill.) was performed according to the manufacturer'sinstructions to confirm the correct constructions. For the finalconstruction of the EN235 SCA gene which is ligatable as an EcoRI—BamHIfragment to P. pastoris vector pHIL-S1, the primer pair5′-CGGAATTCGACGTCGTGATGTCACAG-3′ (SEQ ID NO:19) and5′-CCAGGATCCTATTAACTGGTCTTGTTGGAGACGGTGACTGA-3′ (SEQ ID NO:20) were usedin a PCR reaction.

The designated changes for two and three tandem or overlapping N-linkedglycosylation sites are provided below.

C-terminal of CC49 with double glycosylation sites:

Ser Val Thr Val Ser Asn Lys Thr Asn Ala Thr Ser Stop BamnHI (SEQ IDNO:14)

TCAGTCACCGTCTCCAACAAGACCAATGCTACCTCTTAATAGGATCC (SEQ ID NO:13)

C-terminal of CC49 with triple glycosylation sites:

Ser Val Thr Val Ser Asn Lys Thr Asn Asn Thr Thr Ser Stop BamHI (SEQ IDNO:16)

TCA GTC ACC GTC TCC AAC AAG ACC AAC AAT ACT ACC TCT TAA G GAT CC (SEQ IDNO:15)

The primer pairs used in the PCR reactions for construction of theseexemplary sites are provided below.

Oligonucleotide 5228: 3′PCR primer for cloning two N-linkedglycosylation sites at C-terminal of CC49 into BamHI site of vectorPhilS1:

5′ CCG GGA TCC TAT TAA GAG GTA GCA TTG GTC TTG TTG GAG ACG GTG (SEQ IDNO:21)

Oligonucleotide 5229: 3′PCR primer to put three N-linked glycosylationsites at C-terminal of CC49 into BamHI site of vector PhilS1:

5′ CCG GGA TCC TTA AGA GGT AGT ATT GTT GGT CTT GTT GGA GACGGTG (SEQ IDNO:22)

Olignucleotide 5230: 3′PCR primer to put two N-linked glycosylationsites at C-terminal of CC49 into EcoRi site of vector pPic9:

5′ CCG GAA TTC TAT TAA GAG GTA GCA TTG GTC TTG TTG GAG ACG GTG (SEQ IDNO:23)

Expression of SCA in Pichia pastoris. The Pichia expression vectorpHIL-S1 (Invitrogen Corporation) was used for expression of both theunmodified and glycosylated SCA. This vector provides a signal sequencederived from the yeast gene PH01 which is fused to the gene of interest.The fusion point is at an EcoRI site. After signal processing, thepredicted N-terminal sequence of the SCA proteins will be REFD—where thenormal N-terminus D (in italics) is preceded by three amino acids. Allcloning and expression procedures for production of SCA in Pichia werecarried out as described in the “Pichia Expression Kit InstructionManual” from Invitrogen Corporation, San Diego, Calif. (Cat. No.K1710-01; 1994). Transformation of P. pastoris GS115 with thepHIL-S1/SCA vectors was performed by the spheroplast transformationprocedure followed by isolation of His⁺ and Mut⁻ phenotypes. Growthprotocols in BMGY and BMMY were also performed as described in theInvitrogen manual. After 48 hours of growth in the BMMY medium at 30°C., the induced culture supernatants were collected followingcentrifugation.

SDS-PAGE. Polyacrylamide gel electrophoresis in the presence of SDS wasperformed using pre-cast 4-20% slab gels from Novex Corporation (SanDiego, Calif.) according to the manufacturer's instructions. Proteinbands were visualized by staining with Coomassie Blue. Area quantitationof stained bands was performed using a Molecular Dynamics PD-SI laserscanner.

Western analysis. Immunoblotting procedures for transfer of proteinsfrom gels to nitrocellulose membranes by the semi-dry method wasperformed as described in Harlow, E., & Lane, D., Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., (1988). Blot development was also performed according tothe procedures in this manual. Briefly, the blotted membranes wereblocked in 1% BSA blocking reagent in PBS at room temperature for 2 hr;washed 3× with PBS; and incubated with 3% BSA in PBS with a 1:1,000dilution of rabbit anti-CC49/218 SCA antibody at 4° C. overnight. Next,a 3% BSA in PBS solution containing a 1:1000 dilution of horseradishperoxidase conjugated goat anti-rabbit IgG was used in a 1 hr incubationat room temperature. After washing with PBS, the membranes weredeveloped with TMBM-500 (MOSS, Inc.) at room temperature for 1 min.

Mucin-Sepharose chromatography. The protocol for preparation ofantigen-coupled cyanogen bromide-activated beads is described in Harlow,E., & Lane, D., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1988). CNBr-activatedSepharose 4B (Sigma Corporation, Cat. No. C9142) was coupled to bovinesubmaxillary mucin (Sigma Corporation, Cat. No. M4503). Five mg of mucinwas dissolved in 1 ml of 0.1 M NaHCO₃, 0.5 M NaCl, pH 8.3. A 2.5 galiquot of CNBr-activated resin was swollen in 1 mM HCl. The mucinsolution was next gently mixed with the resin for 1 hr at 22° C. Afterwashing off unbound mucin with 25 ml of the above coupling buffer, theSepharose 4B-mucin was transferred into 0.1 M Tris-HCl pH 8.0. The resinwas washed with 0.1 M sodium acetate, pH 4.0, 0.5 M NaCl; and 0.1 MTris-HCl, pH 8.0, 0.5 M NaCl, alternately for three cycles. The gel waspoured into a 10 ml column and washed with 25 ml of 0.1 M Tris-HCl, pH8.0, 0.1 M NaCl. The EN235 Pichia culture supernatant was dialyzedagainst 0.1 M Tris-HCl, pH 7.4, 0.1 M NaCl at 4° C. overnight, thenloaded onto the mucin-Sepharose column. The column was washed with 0.1 MTris-HCl, pH 8.0, 0.1 M NaCl until the OD280=0 (˜50 ml). Elution of thebound SCA proteins was performed by using 10 ml of eluent 1(0.1 M sodiumcitrate, pH 4.0) followed by 10 ml of eluent 2 (8 M urea, 0.1 MTris-HCl, pH 7.4). The bound SCA eluted in eluent 2.

Endoglycosidase digestion. Peptide-N-Glycosidase F and Endo-glycosidaseH were obtained from Oxford GlycoSystems (Rosedale, N.Y.) and usedaccording to the accompanying product literature.

Glycoprotein staining. The GlycoTrack™ carbohydrate detection kit (Cat.No. K-050) was purchased from Oxford GlycoSystems (Rosedale, N.Y.) andused according to the manufacturer's instructions.

Binding of glyco-SCA to Con A Sepharose. Con A Sepharose was obtainedfrom Pharmacia Biotech (Cat. No. 17-0440-03) and was used according tothe manufacturer's instructions. One ml of resin in binding buffer (20mM Tris-HCl, pH 7.4, 0.5 M NaCl) was incubated with 50 μl of dialyzedEN235 culture supernatant for 30 min at 22° C. The beads were pelletedby microcentrifugation, and the supernatant was removed. Elution of thebound glyco-SCA was performed by washing the resin with binding buffercontaining 0.2 M alpha-D-methylmannoside.

ELISA for SCA binding activity. Immunoassay procedures were performedusing modifications of protocols from Harlow, E., & Lane, D.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., (1988). Direct binding assays were performedand a dose response curve was constructed. Bovine submaxillary mucin(250 ng per 100 μl well) antigen was used to coat microtiter plate wells(MaxiSorp, Nunc, VWR Scientific, Boston, Mass.). The EN225 or purifiedCC49/218 SCA proteins were diluted serially in PBS containing 1% BSA andincubated in the coated wells at 22° C. for 1 hr. After the plate waswashed with PBS containing 0.05% Tween 20 (PBS-T), the bound SCA wasdetected by a 1 hr incubation with a secondary antibody (mouseanti-CC49/218), followed by a PBS-T wash, and a 1 hr incubation with analkaline phosphatase conjugated rabbit anti-mouse IgG antibody. (ForFIG. 10, horseradish peroxidase conjugated goat anti-rabbit IgG wasused.) Signal generation was performed using PNPP as described in Harlowand Lane (page 597). The plate was read at 405 nm using a MolecularDevices (Sunnyvale, Calif.) plate reader.

Example 2 Synthesis of Asn-linked Glycosylation Sequences in Other SCAs

Using the methods described in Example 1, oligonucleotide-directedmutagenesis is employed to create Asn-linked glycosylation consensussequences in the identified loop regions of a Kabat consensusV_(k)I/218/V_(H)III SCA, C6.5/218 SCA, and A33/218 SCA, as shown inFIGS. 6-8, respectively (i.e., (1) two V_(L) changes; (2) two V_(H)changes; (3) one linker change; (4) one C-terminal change; or (5)combinations thereof). In V_(k)I/218/V_(H)III SCA and C6.5/218 SCA,proline residues flanking the tripeptide sequence in the +3 position arechanged to alanines, as recommended by the compilation of Gavel, Y., andvon Heijne, G., Protein Engng. 3:433-442 (1990). Amino acid assignmentsof the Kabat consensus V_(K)I/218/V_(H)III SCA and A33/218 SCA areaccording to Kabat et al., Sequences of Proteins of ImmunologicalInterest, pp. 108 & 331, 5th ed., U.S. Dept. Health and Human Services,Bethesda, Md. (1991), where the assigned amino acid residue at aposition is the most commonly occurring amino acid at that position.Amino acid assignments of the wild-type C6.5 variable domains areaccording to Schier, R., et al., J. Mol. Biol. 255:28-43 (1996).

As described in the “Materials and Methods” section of Example 1, themutated SCAs are individually ligated into the Pichia transfer plasmidpHIL-S1 (Invitrogen Corp.) and transformed into Pichia pastoris.Detailed protocols for these procedures are presented in the PichiaExpression Kit Instruction Manual Cat. No. X1710-01 (1994) fromInvitrogen Corporation. The SCA variants are placed behind a yeastsignal sequence in these constructions and the integrated SCA in theyeast transformants are tested for secretion of the SCA protein orglycoprotein products. Evaluation of expression is done by Coomassiestaining of SDS-PAGE gels. Further tests can be done to confirmexpression of glycosylated SCAs as described in Example 1.

Example 3 Purification of Glyco-CC49

The Pichia cells were harvested from a fermenter and centrifuged at 5000rpm for 40 minutes. The clarified medium was collected and filteredthrough a 0.22 um filter. The sample was dialyzed against water with amembrane of molecular weight cut off of 3500 to a final conductivity ofless than 1 mS. A cation exchange column (Poros-HS) was equilibratedwith 15 mM Tris-Acetate at pH 6.15. The sample was adjusted to pH 6.2and loaded onto the column. Glyco-CC49 was then eluted out with a saltconcentration of 0.15 M NaCl in Tris-acetate buffer pH 7.4. It was thenpassed through a Poros-HQ column equilibrated with 0.15 M NaCl.Tris-acetate buffer pH 7.4. The flow through material was then processedon a size exclusion column (Pharmacia, Superdex-75). The fractionscorresponding to a molecular weight of 25-35 kDa were collected.

Example 4 Site Specific PEGylation of Double Site-Single Chain gCC49/2by PEG-Hydrazide

Purified gCC49/2 (EN279) was concentrated to 2 mg/ml in Tris-HCl, pH7,0.1M NaCl and then passed through a size exclusion column (Superdex-75)into PEGYLATION buffer (0.1 M acetate, pH 5.5). Sodium periodate wasadded so that the final concentration was 10 mM. The sample was oxidizedfor 1 hour in the dark at room temperature. At the end of the reaction,glycerol was added to a final concentration of 5% and the sample wasloaded onto a size exclusion column to remove unreacted sodiumperiodate. The protein was then concentrated to 2 mg/ml.

PEG-hydrazide (5000 molecular weight) (Shearwater) dissolved in the samebuffer was added to the protein at a molar ratio of PEG:protein =100:1.The reaction was allowed to proceed at 37° C. with shaking for 2.5hours.

Sodium borohydride made in PBS (phosphate buffered saline) was added tothe reaction mixture to a final concentration of 10 mM. It was thenstirred for 10 minutes at room temperature.

The reaction product was then analyzed on SDS-polyacrylamide gel and thesize exclusion chromatography analysis. SEC chromatography analysis ofthe reaction mixture showed the appearance of high molecular weightpeaks in addition to the low molecular weight non-glycosylated peakwhich was the only peak before PEGYLATION (FIG. 11A). FIGS. 11B and 12indicate that the PEGYLATION reaction is specific for the carbohydratemoiety and does not affect the single chain antigen binding moleculethat contains no carbohydrate.

Example 5 Site Specific PEGylation of Triple Site-Single Chain gCC49/3by PEG-Hydrazide

The purified gCC49/3 (EN280) was concentrated to 2 mg/ml in 10 mM sodiumacetate buffer pH 7. Just before the PEGYLATION reaction, the pH wasadjusted to 5.5 by adding 1/10 volume of 1 M sodium acetate pH 5.5.Fresh sodium periodate prepared in acetate buffer pH 5.5 was then addedto a final concentration of 10 mM. The protein was then oxidized for 1hour in the dark at room temperature. At the end of the reaction, thesample was loaded onto a size exclusion column to remove unreactedsodium periodate. The protein was then concentrated to 2.5 mg/ml.

PEG-hydrazide (5000 molecular weight) (Shearwater) made in the samebuffer, at a molar excess of 140-fold over that of the protein, wasadded to the protein. The reaction was allowed to proceed at roomtemperature (25° C.) with shaking for 2.5 hours.

Sodium borohydride in PBS was added to the reaction mixture to a finalconcentration of 10 mM and the mixture was stirred for 10 min at roomtemperature.

The reaction product was fractionated on a size exclusion column. Thepurified products were than analyzed by SDS-PAGE (FIG. 13). SDS-PAGEanalysis of glycosylated CC49/3, which either was unmodified or modifiedwith PEG, shows that the PEG modified, glycosylated CC49/3 has a muchhigher molecular weight than the unmodified species. This indicates thatglycosylated CC49/3 is also capable of being PEGylated.

Example 6 Circulation Life of Glyco-SCA and PEG-Glyco-SCA

Sixty μg of glycosylated SCA purified from Pichia pastoris strain EN280,or sixty micrograms of this Glyco-SCA which was PEG-modified, wereinjected intravenously at time 0 into ICR (CD-1) female mice (Harlan-25g, 7-8 weeks old). Mice were bled at the time points indicated in FIG.14. The percent retention in plasma was quantitated by ELISA methods.For the PEG-modified conjugate, Glyco-CC49/218 SCA was conjugated toPEG-hydrazide of molecular mass 5,000 (the protocol is described inZalipsky, S., et al., PCT WO 92/16555, which disclosure is incorporatedherein by reference). The average PEG:SCA molar ratio in the testedPEG-Glyco-SCA conjugate was approximately 4:1.

Example 7 Pharmacokinetics of Plasma Retention of SCA and PEG-SCA

Sixty μg of CC49/218 SCA protein or 60 μg of PEG-modified SCA proteinwere injected intravenously at time 0 into ICR (CD-1) female mice(Harlan-25 g, 7-8 weeks old). Mice were bled at the time pointsindicated in FIG. 15. The percent retention in plasma was quantitated byELISA methods. For the PEG-modified conjugate, CC49/218 SCA wasconjugated to SC-PEG of molecular mass 20,000 (the protocol is describedin U.S. Pat. No. 5,122,614, which disclosure is incorporated herein byreference). The average PEG:SCA molar ratio in the tested PEG-SCAconjugate was approximately 1:1.

Example 8 Affinity Constant (K_(d)) Determinations of Glycosylated andPEGylated CC49 SCA

Competition ELISA methods were performed, as according to Harlow andLane, using biotinylated CC49 SCA, to determine the affinity constants(K_(d)). The results are provided below in Table 3.

TABLE 3 Summary of PEG-Glyco-CC49/Triple Site Binding Data Sample K_(d)(nM) PEG Bio-CC49 6.2 none Native-CC49 3.60 none Native GC 7.34 none P1B48.37 low ˜ 3 P1A 51.54 medium Z919 341.00 high > 8

The non-PEGylated CC49 SCA (Native-CC49), the biotinylated CC49 SCA(Bio-CC49), and the Triple-site Glycosylated CC49 SCA (Native GC; EN280)all have quite similar K_(d) values of 3.6 nM, 6.2 nM, and 7.34 nMrespectively. The PEGylated versions of the Triple-site Glyco-SCA showedreduced but substantial mucin-binding affinity. The P1B preparation withapproximately 3 PEG polymers per SCA (EN280) has a K_(d) of 48.37 nM.The P1A preparation with a PEG/SCA (EN280) molar ratio of approximately4-7 shows a K_(d) of 51.54. The Z919 preparation with a PEG/SCA (EN280)molar ratio of >8 gives a K_(d) of 341 nM.

Example 9 SCA Having an Addition of Five Residues Following theC-terminus N-linked Glycosylation Sequence

We have already established that a single tripeptide N-linkedglycosylation sequence adjacent to the C-terminus of the secondpolypeptide followed by just one additional residue gives adequateglycosylation. About 50% of the SCA is glycosylated. Since theliterature indicates that C-terninal N-linked modification is rare, weinvestigated, using the methods previously described, infra, whether theaddition of five tailing residues, rather than just one, would promotemore efficient overall core glycosylation. The result of the Westernblot (FIG. 16, EN292) for this variant shows that ≧90% of the SCA ismodified with apparent core glycosylation, with no evidence ofhyperglycosylation. This provides an improvement for production ofGlyco-SCA with N-linked core glycosylation. The gene sequence used forthis variant is as follows.

Pichia Strain Number EN292

Ser Val Thr Val Ser Asn Lys Thr Ser Gly Ser Thr Ser End (SEQ ID NO:25)

TCA GTC ACC GTC TCC AAC AAG ACC TCT GGT TCC ACC TCT TAA (SEQ ID NO:24)

Five total amino acids follow the initial N-linked tripeptideglycosylation sequence which is underlined. The last five residues ofthe unmodified CC49 SCA are indicated in bold type.

Example 10 SCA Having Three Tandem N-linked Glycosylation SequencesAdjacent to the C-terminus

We have already shown that a triple-sequence variant adjacent to theC-termninus (containing two tandem plus one overlapping site) producesnearly complete overall glycosylation with predominanthyperglycosylation. We wished to next investigate whether a purely threetandem site version of this would give similar results. As shown in theWestern Blot (FIG. 16, EN293), the three tandem sequence versionproduced complete modification which is exclusively in thehyperglycosylation category. This appears to be a more homogeneousproduct than the previous triple-site version by Western. However, ourinitial results indicate that overall expression is reduced. Hence, thismay be a potential improvement in the triple sequence approach ifexpression can be improved. The gene sequence for this variant is shownbelow with notations as in Example 9.

Pichia Strain Number EN293

Ser Val Thr Val Ser Asn Lys Thr Asn Ala Thr Asn Ala Thr Ser End (SEQ IDNO:27)

TCA GTC ACC GTC TCC AAC AAG ACC AAT GCT ACC AAT GCC ACT TCT TAA (SEQ IDNO:26)

Example 11 SCA Having Two Overlapping Glycosylation Sequences Adjacentto the C-terminus

We have already shown that an SCA having two tandem N-linked tripeptidesequences adjacent to the C-terminus of the second polypeptide produce˜70-90% total modification with both two-site core glycosylation andsome hyperglycosylation present. Additionally, a small amount of theunmodified SCA is observed. We next investigate here a two tripeptidesequence version adjacent to the C-terminus which has overlappingtripeptide sequences rather than tandem sequences. As seen on theWestern Blot (FIG. 16, EN294), this variant produced no unmodified SCA,but produced predominately hyperglycosylated SCA with some apparenttwo-site core modification as judged by the molecular weights of theseproducts on the SDS-PAGE gel used for the Western blot. This resultshows that the phenomenon of “hyperglycosylation” in Pichia can beefficiently induced in Pichia by the minimum overlapping two-sitesequence Asn-Asn-Thr-Thr placed just one residue from the C-terminus. Incontrast, a similar single-sequence version shows relatively lessoverall modification and essentially no detectable hyperglycosylation.The gene sequence for this variant is shown below with notations as inExample 9.

Pichia Strain EN294

Ser Val Thr Val Ser Ser Lys Thr Asn Asn Thr Thr Ser End (SEQ ID NO:29)

TCA GTC ACC GTC TCC TCT AAG ACC AAC AAT ACT ACC TCT TAA (SEQ ID NO:28)

Example 12 SCA Having Three N-Linked Glycosylation Sequence in theLinker Region

We have previously found evidence of inefficient glycosylation in asingle N-linked tripeptide sequence engineered into the 218 linker ofCC49/218 SCA. We further investigated the glycosylation of a tripletripeptide sequence inserted into the SmaI site of the 218 linker. TheWestern blot (FIG. 17, EN290) showed that a heterogeneous mixture ofunmodified, core modified, and hyperglycosylated SCA was observed.Preliminary ELISA confirms the retention of mucin-binding activity.Hence this result shows that active Glyco-SCA can be produced fromPichia with the N-linked site(s) in the linker. However, our C-terminaldata look cleaner overall. The gene sequence of this variant is asfollows with the flanking 218 linker sequence opened at the SmaI siteshown in bold.

Pichia Strain EN290

Pro Asn Lys Thr Asn Asn Thr Thr Gly-(SEQ ID NO:31)

CCC AAC AAG ACC AAC AAT ACT ACC GGG-(SEQ ID NO:30)

Example 13 SCA Having Six N-linked Glycosylation Sequences in the LinkerRegion

We also inserted six N-linked sequences into the SmaI site of the 218linker. A Western blot indicated that expression was poor but overallglycosylation appears complete, although mostly core 1-site. Hence, thisvariant could be of value if expression levels can be improved. Onceagain, it seems that the C-terminal versions may be easier to work with.The gene sequence for this variant is as follows with the flanking 218linker sequence opened at the SmaI site shown in bold.

Pichia Strain EN291

Pro Asn Lys Thr Asn Asn Thr Thr Asn Lys Thr Asn Asn Thr Thr Gly-(SEQ IDNO:33)

CCC AAC AAG ACC AAC AAT ACT ACC AAC AAG ACC AAC AAT ACT ACC GGG-(SEQ IDNO:32)

ELISA results show that each of the variants in Examples 9-13 describedabove maintains mucin-binding specificity. The Coomassie stainedSDS-PAGE gels used for the Western blots were also examined for relativeexpression yields. Pichia vector pHIL-S1 was used as previously for thiswork.

It will be appreciated by those skilled in the art that the inventioncan be performed within a wide range of equivalent parameters ofcomposition, concentrations, modes of administration, and conditionswithout departing from the spirit or scope of the invention or anyembodiment thereof.

All documents, e.g., scientific publications, patents and patentpublications recited herein, are hereby incorporated by reference intheir entirety to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by referencein its entirety.

33 758 base pairs nucleic acid both both cDNA not provided CDS 1..747 1GAC GTC GTG ATG TCA CAG TCT CCA TCC TCC CTA CCT GTG TCA GTT GGC 48 AspVal Val Met Ser Gln Ser Pro Ser Ser Leu Pro Val Ser Val Gly 1 5 10 15GAG AAG GTT ACT TTG AGC TGC AAG TCC AGT CAG AGC CTT TTA TAT AGT 96 GluLys Val Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 GGTAAT CAA AAG AAC TAC TTG GCC TGG TAC CAG CAG AAA CCA GGG CAG 144 Gly AsnGln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 TCT CCTAAA CTG CTG ATT TAC TGG GCA TCC GCT AGG GAA TCT GGG GTC 192 Ser Pro LysLeu Leu Ile Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val 50 55 60 CCT GAT CGCTTC ACA GGC AGT GGA TCT GGG ACA GAT TTC ACT CTC TCC 240 Pro Asp Arg PheThr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser 65 70 75 80 ATC AGC AGTGTG AAG ACT GAA GAC CTG GCA GTT TAT TAC TGT CAG CAG 288 Ile Ser Ser ValLys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 TAT TAT AGC TATCCC CTC ACG TTC GGT GCT GGG ACC AAG CTT GTG CTG 336 Tyr Tyr Ser Tyr ProLeu Thr Phe Gly Ala Gly Thr Lys Leu Val Leu 100 105 110 AAA GGC TCT ACTTCC GGT AGC GGC AAA CCC GGG AGT GGT GAA GGT AGC 384 Lys Gly Ser Thr SerGly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 ACT AAA GGT CAGGTT CAG CTG CAG CAG TCT GAC GCT GAG TTG GTG AAA 432 Thr Lys Gly Gln ValGln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys 130 135 140 CCT GGG GCT TCAGTG AAG ATT TCC TGC AAG GCT TCT GGC TAC ACC TTC 480 Pro Gly Ala Ser ValLys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 145 150 155 160 ACT GAC CATGCA ATT CAC TGG GTG AAA CAG AAC CCT GAA CAG GGC CTG 528 Thr Asp His AlaIle His Trp Val Lys Gln Asn Pro Glu Gln Gly Leu 165 170 175 GAA TGG ATTGGA TAT TTT TCT CCC GGA AAT GAT GAT TTT AAA TAC AAT 576 Glu Trp Ile GlyTyr Phe Ser Pro Gly Asn Asp Asp Phe Lys Tyr Asn 180 185 190 GAG AGG TTCAAG GGC AAG GCC ACA CTG ACT GCA GAC AAA TCC TCC AGC 624 Glu Arg Phe LysGly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 195 200 205 ACT GCC TACGTG CAG CTC AAC AGC CTG ACA TCT GAG GAT TCT GCA GTG 672 Thr Ala Tyr ValGln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val 210 215 220 TAT TTC TGTACA AGA TCC CTG AAT ATG GCC TAC TGG GGT CAA GGA ACC 720 Tyr Phe Cys ThrArg Ser Leu Asn Met Ala Tyr Trp Gly Gln Gly Thr 225 230 235 240 TCA GTCACC GTC TCC AAC AAG ACC AGT TAATAGGATC C 758 Ser Val Thr Val Ser Asn LysThr Ser 245 249 amino acids amino acid linear protein not provided 2 AspVal Val Met Ser Gln Ser Pro Ser Ser Leu Pro Val Ser Val Gly 1 5 10 15Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser 65 70 7580 Ile Ser Ser Val Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 9095 Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Val Leu 100105 110 Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser115 120 125 Thr Lys Gly Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu ValLys 130 135 140 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly TyrThr Phe 145 150 155 160 Thr Asp His Ala Ile His Trp Val Lys Gln Asn ProGlu Gln Gly Leu 165 170 175 Glu Trp Ile Gly Tyr Phe Ser Pro Gly Asn AspAsp Phe Lys Tyr Asn 180 185 190 Glu Arg Phe Lys Gly Lys Ala Thr Leu ThrAla Asp Lys Ser Ser Ser 195 200 205 Thr Ala Tyr Val Gln Leu Asn Ser LeuThr Ser Glu Asp Ser Ala Val 210 215 220 Tyr Phe Cys Thr Arg Ser Leu AsnMet Ala Tyr Trp Gly Gln Gly Thr 225 230 235 240 Ser Val Thr Val Ser AsnLys Thr Ser 245 263 amino acids amino acid single Not Relevant peptidenot provided 3 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala SerVal Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser LeuVal Ser Ile 20 25 30 Ser Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly LysAla Pro Lys 35 40 45 Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly ValPro Ser Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu ThrIle Ser Ser 65 70 75 80 Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys GlnGln Tyr Asn Ser 85 90 95 Leu Pro Glu Trp Thr Phe Gly Gln Gly Thr Lys ValGlu Ile Lys Gly 100 105 110 Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser GlyGlu Gly Ser Thr Lys 115 120 125 Gly Glu Val Gln Leu Val Glu Ser Gly GlyGly Leu Val Gln Pro Gly 130 135 140 Gly Ser Leu Arg Leu Ser Cys Ala AlaSer Gly Phe Thr Phe Ser Ser 145 150 155 160 Tyr Ala Met Ser Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp 165 170 175 Val Ser Val Ile Ser GlyLys Thr Asp Gly Gly Ser Thr Tyr Tyr Ala 180 185 190 Asp Ser Val Lys GlyArg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 195 200 205 Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 210 215 220 Tyr Tyr CysAla Arg Gly Arg Xaa Gly Xaa Ser Leu Ser Gly Xaa Tyr 225 230 235 240 TyrTyr Tyr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 245 250 255Val Ser Ser Asn Lys Thr Ser 260 262 amino acids amino acid single NotRelevant peptide not provided 4 Gln Ser Val Leu Thr Gln Pro Pro Ser ValSer Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly SerSer Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu ProGly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly His Thr Asn Arg Pro AlaGly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala SerLeu Ala Ile Ser Gly Phe Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr TyrCys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Trp Val Phe Gly Gly GlyThr Lys Leu Thr Val Leu Gly Gly 100 105 110 Ser Thr Ser Gly Ser Gly LysPro Gly Ser Gly Glu Gly Ser Thr Lys 115 120 125 Gly Gln Val Gln Leu LeuGln Ser Gly Ala Glu Leu Lys Lys Pro Gly 130 135 140 Glu Ser Leu Lys IleSer Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser 145 150 155 160 Tyr Trp IleAla Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Tyr 165 170 175 Met GlyLeu Ile Tyr Pro Gly Asp Ser Asp Thr Lys Tyr Ser Pro Ser 180 185 190 PheGln Gly Gln Val Thr Ile Ser Val Asp Lys Ser Val Ser Thr Ala 195 200 205Tyr Leu Gln Trp Ser Ser Leu Lys Pro Ser Asp Ser Ala Val Tyr Phe 210 215220 Cys Ala Arg His Asp Val Gly Tyr Cys Ser Ser Ser Asn Cys Ala Lys 225230 235 240 Trp Pro Glu Tyr Phe Gln His Trp Gly Gln Gly Thr Leu Val ThrVal 245 250 255 Ser Ser Asn Lys Thr Ser 260 245 amino acids amino acidsingle Not Relevant peptide not provided 5 Asp Val Val Met Thr Gln SerGln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile ThrCys Lys Ala Ser Gln Asn Val Arg Thr Val 20 25 30 Val Ala Trp Tyr Gln GlnLys Pro Gly Gln Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Leu Ala Ser Asn ArgHis Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr AspPhe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala AspTyr Phe Cys Leu Gln His Trp Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Ser GlyThr Lys Leu Glu Val Lys Gly Ser Thr Ser Gly 100 105 110 Ser Gly Lys ProGly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys 115 120 125 Leu Val GluSer Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys 130 135 140 Leu SerCys Ala Ala Ser Gly Phe Ala Phe Ser Thr Tyr Asp Met Ser 145 150 155 160Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr Ile 165 170175 Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Leu Asp Ser Val Lys Gly Arg 180185 190 Phe Thr Ile Ser Arg Asp Ser Ala Arg Asn Thr Leu Tyr Leu Gln Met195 200 205 Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Ala ProThr 210 215 220 Thr Val Val Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu ValThr Val 225 230 235 240 Ser Asn Lys Thr Ser 245 12 amino acids aminoacid single Not Relevant peptide not provided 6 Gly Lys Ser Ser Gly SerGly Ser Glu Ser Lys Ser 1 5 10 14 amino acids amino acid single NotRelevant peptide not provided 7 Gly Ser Thr Ser Gly Ser Gly Lys Ser SerGlu Gly Lys Gly 1 5 10 18 amino acids amino acid single Not Relevantpeptide not provided 8 Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu GlySer Gly Ser Thr 1 5 10 15 Lys Gly 12 amino acids amino acid single NotRelevant peptide not provided 9 Gly Ser Thr Ser Gly Lys Pro Ser Glu GlyLys Gly 1 5 10 18 amino acids amino acid single Not Relevant peptide notprovided 10 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly SerThr 1 5 10 15 Lys Gly 38 base pairs nucleic acid both both cDNA notprovided CDS 1..27 11 TCA GTC ACC GTC TCC AAC AAG ACC AGT TAATAGGATC C38 Ser Val Thr Val Ser Asn Lys Thr Ser 250 255 9 amino acids amino acidlinear protein not provided 12 Ser Val Thr Val Ser Asn Lys Thr Ser 1 547 base pairs nucleic acid both both cDNA not provided CDS 1..36 13 TCAGTC ACC GTC TCC AAC AAG ACC AAT GCT ACC TCT TAATAGGATC 46 Ser Val ThrVal Ser Asn Lys Thr Asn Ala Thr Ser 10 15 20 C 47 12 amino acids aminoacid linear protein not provided 14 Ser Val Thr Val Ser Asn Lys Thr AsnAla Thr Ser 1 5 10 48 base pairs nucleic acid both both cDNA notprovided CDS 1..39 15 TCA GTC ACC GTC TCC AAC AAG ACC AAC AAT ACT ACCTCT TAAGGATCC 48 Ser Val Thr Val Ser Asn Lys Thr Asn Asn Thr Thr Ser 1520 25 13 amino acids amino acid linear protein not provided 16 Ser ValThr Val Ser Asn Lys Thr Asn Asn Thr Thr Ser 1 5 10 6 amino acids aminoacid single Not Relevant peptide not provided 17 Asn Lys Thr Asn Ala Thr1 5 7 amino acids amino acid single Not Relevant peptide not provided 18Asn Lys Thr Asn Asn Thr Thr 1 5 26 base pairs nucleic acid both bothcDNA not provided 19 CGGAATTCGA CGTCGTGATG TCACAG 26 41 base pairsnucleic acid both both cDNA not provided 20 CCAGGATCCT ATTAACTGGTCTTGTTGGAG ACGGTGACTG A 41 45 base pairs nucleic acid both both cDNA notprovided 21 CCGGGATCCT ATTAAGAGGT AGCATTGGTC TTGTTGGAGA CGGTG 45 46 basepairs nucleic acid both both cDNA not provided 22 CCGGGATCCT TAAGAGGTAGTATTGTTGGT CTTGTTGGAG ACGGTG 46 45 base pairs nucleic acid both bothcDNA not provided 23 CCGGAATTCT ATTAAGAGGT AGCATTGGTC TTGTTGGAGA CGGTG45 42 base pairs nucleic acid both both cDNA not provided CDS 1..39 24TCA GTC ACC GTC TCC AAC AAG ACC TCT GGT TCC ACC TCT TAA 42 Ser Val ThrVal Ser Asn Lys Thr Ser Gly Ser Thr Ser 250 255 260 13 amino acids aminoacid linear protein not provided 25 Ser Val Thr Val Ser Asn Lys Thr SerGly Ser Thr Ser 1 5 10 48 base pairs nucleic acid both both cDNA notprovided CDS 1..45 26 TCA GTC ACC GTC TCC AAC AAG ACC AAT GCT ACC AATGCC ACT TCT 45 Ser Val Thr Val Ser Asn Lys Thr Asn Ala Thr Asn Ala ThrSer 15 20 25 TAA 48 15 amino acids amino acid linear protein notprovided 27 Ser Val Thr Val Ser Asn Lys Thr Asn Ala Thr Asn Ala Thr Ser1 5 10 15 42 base pairs nucleic acid both both cDNA not provided CDS1..39 28 TCA GTC ACC GTC TCC TCT AAG ACC AAC AAT ACT ACC TCT TAA 42 SerVal Thr Val Ser Ser Lys Thr Asn Asn Thr Thr Ser 20 25 13 amino acidsamino acid linear protein not provided 29 Ser Val Thr Val Ser Ser LysThr Asn Asn Thr Thr Ser 1 5 10 27 base pairs nucleic acid both both cDNAnot provided CDS 1..27 30 CCC AAC AAG ACC AAC AAT ACT ACC GGG 27 Pro AsnLys Thr Asn Asn Thr Thr Gly 15 20 9 amino acids amino acid linearprotein not provided 31 Pro Asn Lys Thr Asn Asn Thr Thr Gly 1 5 48 basepairs nucleic acid both both cDNA not provided CDS 1..48 32 CCC AAC AAGACC AAC AAT ACT ACC AAC AAG ACC AAC AAT ACT ACC GGG 48 Pro Asn Lys ThrAsn Asn Thr Thr Asn Lys Thr Asn Asn Thr Thr Gly 10 15 20 25 16 aminoacids amino acid linear protein not provided 33 Pro Asn Lys Thr Asn AsnThr Thr Asn Lys Thr Asn Asn Thr Thr Gly 1 5 10 15

What is claimed is:
 1. A single-chain antigen-binding polypeptidecomprising: (a) a first polypeptide comprising the antigen bindingportion of the variable region of an antibody heavy or light chain; (b)a second polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain; (c) a peptidelinker linking said first and second polypeptides (a) and (b) into asingle chain polypeptide having an antigen binding site, wherein saidsingle-chain antigen-binding polypeptide has at least one tripeptideAsn-linked glyscosylation amino acid residue comprising Asn-Xaa-Yaa,wherein Xaa is an amino acid other than proline and Yaa is threonine orserine and said Asn residue is located at a position selected from thegroup consisting of (i) the amino acid positions 11, 12, 13, 14 or 15 ofsaid light chain variable region; (ii) the amino acid positions 77, 78,or 79 of said light chain variable region; (iii) the amino acidpositions 11, 12, 13, 14 or 15 of said heavy chain variable region; (iv)the amino acid positions 82B, 82C or 83 of said heavy chain variableregion; (v) the amino acid position 2 of said peptide linker; (vi) anamino acid position in the amino acid sequence which is adjacent to theC-terminus amino acid residue of said second polypeptide (b); and (vii)combinations thereof.
 2. The polypeptide of claim 1, which has at leasttwo said tripeptide glycosylation amino acid residues in tandem suchthat the Asn residues are separated by two amino acid residues.
 3. Thepolypeptide of claim 2, which has three said tripeptide glycosylationamino acid residues in tandem such that the Asn residues are separatedby two amino acid residues.
 4. The polypeptide of claim 1, which has atleast one set of two overlapping said tripeptide glycosylation aminoacid residues such that the Asn residues are adjacent.
 5. Thepolypeptide of claim 1, which has (A) at least two said tripeptideglycosylation amino acid residues in tandem such that the Asn residuesare separated by two amino acid residues and (B) at least one set of twooverlapping said tripeptide glycosylation amino acid residues such thatthe Asn residues are adjacent.
 6. The polypeptide of claim 5, which hasat least two sets of two tandem said tripeptide glycosylation amino acidresidues and at least two sets of two overlapping said tripeptideglycosylation amino acid residues.
 7. The polypeptide of claim 1,wherein said Asn residue is located at a position selected from thegroup consisting of (i) the amino acid position 12 of the light chainvariable region; (ii) the amino acid position 77 of the light chainvariable region; (iii) the amino acid position 13 of the heavy chainvariable region; (iv) the amino acid position 82B of the heavy chainvariable region; (v) an amino acid position in the amino acid sequencewhich is adjacent to the C-terminus amino acid residue of said secondpolypeptide (b); and (vi) combinations thereof.
 8. The polypeptide ofclaim 1, wherein said first polypeptide (a) comprises the antigenbinding portion of the variable region of an antibody light chain andsaid second polypeptide (b) comprises the antigen binding portion of thevariable region of an antibody heavy chain.
 9. The polypeptide of claim1, wherein the C-terminus of said second polypeptide (b) is the nativeC-terminus of said second polypeptide (b).
 10. The polypeptide of claim1, wherein the C-terminus of said second polypeptide (b) comprises adeletion of one or plurality of amino acid residue(s) from the nativeC-terminus of said second polypeptide (b), such that the remainingN-terminus amino acid residues of the second polypeptide are sufficientfor the glycosylated single-chain antigen-binding polypeptide to becapable of binding an antigen.
 11. The polypeptide of claim 1, whereinthe C-terminus of said second polypeptide (b) comprises an addition ofone or plurality of amino acid residue(s) to the native C-terminus ofsaid second polypeptide (b), such that the glycosylated single-chainantigen binding polypeptide is capable of binding an antigen.
 12. Thepolypeptide of claim 9, wherein said Asn residue of said glycosylationamino acid residues is located adjacent to said native C-terminus ofsaid second polypeptide (b) and said glycosylation amino acid residuesare followed by at least one additional amino acid residue.
 13. Thepolypeptide of claim 12, wherein said glycosylation sequence is followedby 5 additional amino acid residues.
 14. The polypeptide of claim 1,wherein said Asn residue of said tripeptide glycosylation amino acidresidues is attached to a carbohydrate moiety.
 15. The polypeptide ofclaim 14, wherein said carbohydrate moiety is conjugated to one orplurality of peptide, lipid, nucleic acid, drug, toxin, chelator, boronaddend or detectable label molecules.
 16. The polypeptide of claim 14,wherein said carbohydrate moiety is conjugated to a carrier having oneor plurality of peptide, lipid, nucleic acid, drug, toxin, chelator,boron addend or detectable label molecules bound to said carrier. 17.The polypeptide of claim 14, wherein said carbohydrate moiety isconjugated to a polyalkylene oxide moiety.
 18. The polypeptide of claim17, wherein said polyalkylene oxide moiety is conjugated to one orplurality of peptide, lipid, nucleic acid, drug, toxin, chelator, boronaddend or detectable label molecules.
 19. The polypeptide of claim 17,wherein said polyalkylene oxide moiety is conjugated to a carrier havingone or plurality of peptide, lipid, nucleic acid, drug, toxin, chelator,boron addend or detectable label molecules bound to said carrier. 20.The polypeptide of claim 10, wherein said Asn residue of saidglycosylation amino acid residues is located adjacent to said C-terminusof said second polypeptide (b) and said glycosylation amino acidresidues is followed by at least one additional amino acid residue. 21.The polypeptide of claim 11, wherein said Asn residue of saidglycosylation amino acid residues is located adjacent to said C-terminusof said second polypeptide (b) and said glycosylation amino acidresidues is followed by at least one additional amino acid residue. 22.The polypeptide of claim 20, wherein said glycosylation amino acidresidues is followed by 5 additional amino acid residues.
 23. Thepolypeptide of claim 21, wherein said glycosylation amino acid residuesis followed by 5 additional amino acid residues.
 24. The polypeptide ofclaim 1, wherein said Asn residue is located at the amino acid position11, 12, 13, 14 or 15 of said light chain variable region.
 25. Thepolypeptide of claim 1, wherein said Asn residue is located at the aminoacid position 77, 78 or 79 of said light chain variable region.
 26. Thepolypeptide of claim 1, wherein said Asn residue is located at the aminoacid position 11, 12, 13, 14 or 15 of said heavy chain variable region.27. The polypeptide of claim 1, wherein said Asn residue is located atthe amino acid position 82B, 82C or 83 of said heavy chain variableregion.
 28. The polypeptide of claim 1, wherein said Asn residue islocated at an amino acid position which is adjacent to the C-terminus ofsaid second polypeptide (b).
 29. The polypeptide of claim 7, whereinsaid Asn residue is located at an amino acid position 12 of the lightchain variable region.
 30. The polypeptide of claim 7, wherein said Asnresidue is located at an amino acid position 77 of the light chainvariable region.
 31. The polypeptide of claim 7, wherein said Asnresidue is located at an amino acid position 13 of the heavy chainvariable region.
 32. The polypeptide of claim 7, wherein said Asnresidue is located at an amino acid position 82B of the heavy chainvariable region.
 33. A single-chain antigen-binding polypeptide,comprising: (a) a first polypeptide comprising the antigen bindingportion of the variable region of an antibody heavy or light chain; (b)a second polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain; and (c) a peptidelinker linking said first and second polypeptides (a) and (b) into asingle chain polypeptide having an antigen binding site, wherein saidsingle-chain antigen-binding polypeptide has at least one set of twooverlapping tripeptide Asn-linked glycosylation amino acid residuescomprising Asn-Asn-Xaa-Yaa, wherein both Xaa and Yaa are threonine orserine and the first Asn residue is located at any amino acid positionof said peptide linker.